Cestrum yellow leaf curling virus promoters

ABSTRACT

This invention describes novel DNA sequences that function as promoters of transcription of associated nucleotide sequences. More specifically, this invention describes DNA sequences conferring constitutive expression to an associated nucleotide sequence. The invention also describes recombinant sequences containing such promoter sequences. The said recombinant DNA sequences may be used to create transgenic plants, but especially plants expressing a nucleotide sequence of interest at all times and in most tissues and organs.

[0001] The present invention relates to novel DNA sequences that function as promoters of transcription of associated nucleotide sequences in plants. More specifically, this invention relates to novel promoters that confer constitutive expression to an associated nucleotide sequence of interest.

[0002] In the field of agriculture there exists a continuous desire to modify plants according to one's needs. One way to accomplish this is by using modern genetic engineering techniques. For example, by introducing a gene of interest into a plant, the plant can be specifically modified to express a desirable phenotypic trait. For this, plants are transformed most commonly with a heterologous gene comprising a promoter region, a coding region and a termination region. When genetically engineering a heterologous gene for expression in plants, the selection of a promoter is a critical factor. While it may be desirable to express certain genes only in response to particular stimuli or confined to specific cells or tissues, other genes are more desirably expressed constitutively, i.e. throughout the plant at all times and in most tissues and organs. In the past, the 35S promoter from Cauliflower Mosaic Virus (CaMV 35S promoter) has been widely used for constitutive expression of heterologous genes in plants. There are, however, occasions where it is desirable to use alternative promoters. Therefore, it is a major objective of the present invention to provide such alternative promoters for expression of a nucleotide sequence of interest in plants. The invention also provides recombinant DNA molecules, expression vectors and transgenic plants comprising the promoters of the present invention.

[0003] The present invention thus provides:

[0004] a DNA sequence capable of driving expression of an associated nucleotide sequence, wherein said DNA sequence is obtainable from the genome of Cestrum yellow leaf curling virus (CmYLCV). Preferred is a DNA sequence which is obtainable from the CmYLCV full-length transcript promoter and comprises the nucleotide sequence depicted in SEQ ID NO:1.

[0005] In particular, DNA sequences are provided, wherein

[0006] said DNA sequence comprises the nucleotide sequence depicted in SEQ ID NO:2

[0007] said DNA sequence comprises the nucleotide sequence depicted in SEQ ID NO:3

[0008] said DNA sequence comprises the nucleotide sequence depicted in SEQ ID NO:4

[0009] said DNA sequence comprises the nucleotide sequence depicted in SEQ ID NO:5

[0010] said DNA sequence comprises the nucleotide sequence depicted in SEQ ID NO:6

[0011] said DNA sequence hybridizes under stringent conditions to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, in particular wherein the DNA sequence as mentioned hereinbefore comprises the nucleotide sequence depicted in SEQ ID NO:19 or SEQ ID NO:20.

[0012] The invention further provides DNA sequences comprising a consecutive stretch of at least 50 nt, preferably of between about 400 bases and about 650 bases, more preferably of between about 200 bases and about 400 bases and most preferably of about 350 bases in length of SEQ ID NO:1, wherein said DNA sequences are capable of driving expression of an associated nucleotide sequence.

[0013] In a particular embodiment of the invention said consecutive stretch of at least 50 nt, preferably of between about 400 bases and about 650 bases, more preferably of between about 200 bases and about 400 bases and most preferably of about 350 bases in length has at least 75%, preferably 80%, more preferably 90% and most preferably 95% sequence identity with a corresponding consecutive stretch of at least 50 nt, preferably of between about 400 bases and about 650 bases, more preferably of between about 200 bases and about 400 bases and most preferably of about 350 bases in length of SEQ ID NO:1.

[0014] The invention further provides recombinant DNA molecules comprising a full-length transcript promoter region isolated from Cestrum yellow leaf curling virus. In addition, the invention provides recombinant DNA molecules and DNA expression cassettes comprising a DNA sequence according to the invention operably linked to a nucleotide sequence of interest. The invention also provides DNA expression vectors comprising said recombinant DNA and expression cassettes, respectively.

[0015] In particular, recombinant DNA molecules and DNA expression cassettes are provided wherein the nucleotide sequence of interest comprises a coding sequence, particularly wherein

[0016] the coding sequence encodes a desirable phenotypic trait

[0017] the coding sequence encodes a selectable or screenable marker gene

[0018] the coding sequence encodes a protein conferring antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, improved nutritional value, improved performance in an industrial process or altered reproductive capability

[0019] the coding sequence encodes a protein which confers a positive selective advantage to cells that have been transformed with said coding sequence

[0020] the coding sequence encodes a protein which confers a metabolic advantage to cells that have been transformed with said coding sequence consisting of being able to metabolize a compound, wherein said compound is mannose or xylose or a derivative or a precursor of these, or a substrate of the protein, or is capable of being metabolized by cells transformed with said coding sequence into such a substrate

[0021] the coding sequence encodes an enzyme selected from the group of xyloisomerases, phosphomanno-isomerase, mannose-6-phosphate isomerase, mannose-1-phosphate isomerase, phosphomanno mutase, mannose epimerase, mannose or xylose phosphatase, mannose-6-phosphatase, mannose-1-phosphatase and mannose or xylose permease

[0022] the coding sequence encodes a phosphomanno isomerase

[0023] the coding region is non-translatable

[0024] the non-translatable coding region is from a viral gene, in particular from TSWV, more particularly from the TSWV NP gene

[0025] the coding sequence encodes commercially valuable enzymes or metabolites in the plant

[0026] the coding sequence is in antisense orientation

[0027] The invention also provides DNA expression vectors comprising a DNA sequence or a recombinant DNA molecule as mentioned hereinbefore. In a particular embodiment of the invention, said DNA expression vector is pNOV2819 or pNOV2819. Further are provided DNA expression vectors comprising a first DNA sequence according to the invention operably linked to a nucleotide sequence of interest, and a second DNA sequence according to the invention operably linked to a nucleotide sequence of interest. In a specific embodiment of the invention the DNA expression vectors as mentioned hereinbefore are capable of altering the expression of a viral genome.

[0028] In an even more specific embodiment, the DNA expression vector as mentioned hereinbefore comprises a first DNA sequence capable of expressing in a cell a sense RNA fragment of said viral genome or portion thereof and a second DNA sequence capable of expressing in a cell an antisense RNA fragment of said viral genome or portion thereof, wherein said sense RNA fragment and said antisense RNA fragment are capable of forming a double-stranded RNA.

[0029] Expression vectors as mentioned hereinbefore are provided wherein

[0030] said virus is selected from the group consisting of tospoviruses, potyviruses, potexviruses, tobamoviruses, luteoviruses, cucumoviruses, bromoviruses, closteorviruses, tombusviruses and furoviruses

[0031] said DNA sequences comprises a nucleotide sequence derived from a viral coat protein gene, a viral nucleocapsid protein gene, a viral replicase gene, or a viral movement protein gene or portions thereof

[0032] said DNA sequence is derived from tomato spotted wilt virus (TSWV)

[0033] said DNA is derived from a nucleocapsid protein gene

[0034] The invention further provides

[0035] host cells stably transformed with a DNA sequence, a recombinant DNA molecule or a DNA expression vector according to the invention. In particular, wherein

[0036] the host cell is a bacterium

[0037] the host cell is a plant cell

[0038] the host cell is a plant cell selected from the group consisting of rice, maize, wheat, barley, rye, sweet potato, sweet corn, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar-beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, potato, eggplant, cucumber, Arabidopsis thaliana, and woody plants such as coniferous and deciduous trees, but particularly rice, maize, wheat, barley, cabbage, cauliflower, pepper, squash, melon, soybean, tomato, sugar-beet, sunflower or cotton, rice, maize, wheat, Sorghum bicolor, orchardgrass, sugar beet and soybean cells

[0039] the host cell is a plant cell from a dicotyledonous plant

[0040] the host cell is a plant cell from a dicotyledonous plant selected from the group consisting of soybean, cotton, tobacco, sugar beet and oilseed rape

[0041] the host cell is a plant cell from a monocotyledonous plant

[0042] the host cell is a plant cell from a monocotyledonous plant selected from the group consisting of maize, wheat, sorghum, rye, oats, turf grass, rice, and barley.

[0043] In addition, plants and the progeny thereof including seeds are provided stably transformed with a DNA sequence, a recombinant DNA molecule or a DNA expression vector according to the invention. In particular, wherein

[0044] the plant is selected from the group consisting of rice, maize, wheat, barley, rye, sweet potato, sweet corn, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar-beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, potato, eggplant, cucumber, Arabidopsis thaliana, and woody plants such as coniferous and deciduous trees, but particularly rice, maize, wheat, barley, cabbage, cauliflower, pepper, squash, melon, soybean, tomato, sugar-beet, sunflower or cotton, rice, maize, wheat, Sorghum bicolor, orchardgrass, sugar beet and soybean.

[0045] The present invention further discloses

[0046] the use of the DNA sequence according to the invention to express a nucleotide sequence of interest

[0047] a method of producing a DNA sequence according to the invention, wherein the DNA is produced by a polymerase chain reaction wherein at least one oligonucleotide used comprises a sequence of nucleotides which represents a consecutive stretch of 15, 18, 20, 22, 24 or more base pairs of SEQ ID NO:1.

[0048] In order to ensure a clear and consistent understanding of the specification and the claims, the following definitions are provided:

[0049] CmYLCV: Cestrum yellow leaf curling virus.

[0050] DNA shuffling: DNA shuffling is a method to rapidly, easily and efficiently introduce rearrangements, preferably randomly, in a DNA molecule or to generate exchanges of DNA sequences between two or more DNA molecules, preferably randomly. The DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is a non-naturally occurring DNA molecule derived from at least one template DNA molecule.

[0051] Expression: refers to the transcription and/or translation of an endogenous gene or a transgene in plants. In the case of antisense constructs, for example, expression may refer to the transcription of the antisense DNA only.

[0052] Functionally equivalent sequence: refers to a DNA sequence which has promoter activity substantially similar to the CmYLCV full-length transcript promoter or parts thereof and which under stringent hybridizing conditions hybridizes with the said promoter sequences.

[0053] Gene: refers to a coding sequence and associated regulatory sequence wherein the coding sequence is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Examples of regulatory sequences are promoter sequences, 5′- and 3′-untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.

[0054] Gene of interest: refers to any gene which, when transferred to a plant, confers upon the plant a desired characteristic such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, improved nutritional value, improved performance in an industrial process or altered reproductive capability. The “gene of interest” may also be one that is transferred to plants for the production of commercially valuable enzymes or metabolites in the plant.

[0055] Heterologous as used herein means of different natural or of synthetic origin. For example, if a host cell is transformed with a nucleic acid sequence that does not occur in the untransformed host cell, that nucleic acid sequence is said to be heterologous with respect to the host cell. The transforming nucleic acid may comprise a heterologous promoter, heterologous coding sequence, or heterologous termination sequence. Alternatively, the transforming nucleic acid may be completely heterologous or may comprise any possible combination of heterologous and endogenous nucleic acid sequences.

[0056] Leader region: region in a gene between transcription start site and translation start site.

[0057] Marker gene: refers to a gene encoding a selectable or screenable trait.

[0058] Operably linked to/associated with: a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a protein if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence.

[0059] Plant: refers to any plant, particularly to seed plants

[0060] Plant cell: structural and physiological unit of the plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, or a plant organ.

[0061] Plant material: refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, pollen tubes, ovules, embryo sacs, egg cells, zygotes, embryos, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant

[0062] Promoter: refers to a DNA sequence that initiates transcription of an associated DNA sequence. The promoter region may also include elements that act as regulators of gene expression such as activators, enhancers, and/or repressors and may include all or part of the 5′ non-translated leader sequence.

[0063] Recombinant DNA molecule: a combination of DNA sequences that are joined together using recombinant DNA technology.

[0064] Recombinant DNA technology: procedures used to join together DNA sequences as described, for example, in Sambrook et al., 1989, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.

[0065] Screenable marker gene: refers to a gene whose expression does not confer a selective advantage to a transformed cell, but whose expression makes the transformed cell phenotypically distinct from untransformed cells.

[0066] Selectable marker gene: refers to a gene whose expression in a plant cell gives the cell a selective advantage. The selective advantage possessed by the cells transformed with the selectable marker gene may be due to their ability to grow in the presence of a negative selective agent, such as an antibiotic or a herbicide, compared to the growth of non-transformed cells. The selective advantage possessed by the transformed cells, compared to non-transformed cells, may also be due to their enhanced or novel capacity to utilize an added compound as a nutrient, growth factor or energy source. Selectable marker gene also refers to a gene or a combination of genes whose expression in a plant cell in the presence of the selective agent, compared to the absence of the selective agent, has a positive effect on the transformed plant cell and a negative effect on the un-transformed plant cell, for example with respect to growth, and thus gives the transformed plant cell a positive selective advantage.

[0067] Sequence identity: the percentage of sequence identity is determined using computer programs that are based on dynamic programming algorithms. Computer programs that are preferred within the scope of the present invention include the BLAST (Basic Local Alignment Search Tool) search programs designed to explore all of the available sequence databases regardless of whether the query is protein or DNA. Version BLAST 2.0 (Gapped BLAST) of this search tool has been made publicly available on the Internet (currently http://www.ncbi.nlm.nih.gov/BLAST/). It uses a heuristic algorithm, which seeks local as opposed to global alignments and is therefore able to detect relationships among sequences, which share only isolated regions. The scores assigned in a BLAST search have a well-defined statistical interpretation. Said programs are preferably run with optional parameters set to the default values.

[0068] Transformation: refers to the introduction of a nucleic acid into a cell. In particular, it refers to the stable integration of a DNA molecule into the genome of an organism of interest

[0069] TSWV: tomato spotted wilt virus

[0070] The present invention relates to DNA sequences obtainable from the genome of the Cestrum yellow leaf curling virus (CmYLCV). Preferred are DNA sequences which are obtainable from the CmYLCV full-length transcript promoter which are capable of driving expression of an associated nucleotide sequence of interest. DNA sequences comprising functional and/or structural equivalents thereof are also embraced by the invention. The present invention thus relates to DNA sequences that function as promoters of transcription of associated nucleotide sequences. The promoter region may also include elements that act as regulators of gene expression such as activators, enhancers, and/or repressors and may include the 5′ non-translated leader sequence of the transcribed mRNA.

[0071] In a preferred embodiment of the invention, said DNA sequence confers constitutive expression to an associated nucleotide sequence. Constitutive expression means that the nucleotide sequence of interest is expressed at all times and in most tissues and organs. When tested in association with a GUS or CAT reporter gene in transient expression assays, the DNA sequence according to the invention confers high level of expression of the GUS or CAT reporter gene. Thus, the DNA sequence according to the invention is useful for high level expression of an associated nucleotide sequence of interest, which preferably is a coding sequence. It is known to the skilled artisan that the associated coding sequence of interest can be expressed in sense or in antisense orientation. Further, the coding sequence of interest may be of heterologous or homologous origin with respect to the plant to be transformed. In case of a homologous coding sequence, the DNA sequence according to the invention is useful for ectopic expression of said sequence. In one particular embodiment of the invention expression of the coding sequence of interest under control of a DNA sequence according to the invention suppresses its own expression and that of the original copy of the gene by a process called cosuppression.

[0072] The promoters of the present invention can be obtained from Cestrum yellow leaf curling virus (CmYLCV) genomic DNA, which can be isolated, for example, from infected Cestrum parqui plants. CmYLCV-infected Cestrum parqui plants are identified by their leaf mosaic and crinkled leaf malformation. This DNA is then sequenced and analyzed. Sequence comparison with sequences in the database shows that the genomic organization of the CmYLCV is related to the one of other members of the Caulimovirus family. CmYLCV contains 8 open reading frames. Sequence comparison of the CmYLCV gene products shows that gene I product is involved in intracellular movements: gene A encodes a small protein of unknown function, gene B codes for the aphid transmission factor; gene C encodes a virion associated protein; the gene IV is the major capsid protein; gene V codes for a reverse transcriptase and the gene VI activates in trans the translation of viral genes on full-length transcript. Of particular interest is the so-called CmYLCV full-length transcript promoter.

[0073] It is known to the skilled artisan that various regions of the CmYLCV full-length transcript promoter can be used according to the invention. One particular embodiment of the invention is the CmYLCV full-length transcript promoter region shown in SEQ ID NO:1, called the CmpD promoter. This sequence contains 350 bp of CmYLCV full-length transcript promoter and 320 bp of the CmYLCV full-length transcript 5′ non-translated leader sequence. This sequence is obtained by sequencing the CmYLCV genomic DNA. The promoter and leader sequences respectively, are identified by comparison to sequences in the database. A variant of SEQ ID NO:1, called CmpE, wherein the CmYLCV full-length transcript 5′ non-translated leader sequence is 318 bp instead of 320 bp in length, is shown in SEQ ID NO:19.

[0074] One preferred embodiment of the invention is the DNA sequence depicted in SEQ ID NO:2, called the CmpC promoter. The CmpC promoter is a fragment of the sequence shown in SEQ ID NO:1 and contains 346 bp of CmYLCV full-length transcript promoter, corresponding to base 5 to 350 of SEQ ID NO:1. This DNA sequence is obtainable by PCR with genomic DNA from Cestrum yellow leaf curling virus or from Cestrum yellow leaf curling virus-infected Cestrum parqui plants using forward primer Cmp1 (SEQ ID NO:13) and reverse primer CmpC2 (SEQ ID NO:14). The putative TATA-box of the CmpC promoter is located from base 308 to base 315 of SEQ ID NO:2. The CmpC promoter contains at least 3 putative enhancer regions. Enhancer region 1 having the nucleotide sequence CAAT is located from base 232 to base 235 of SEQ ID NO:2, and enhancer region 2 also having the nucleotide sequence CAAT is located from base 243 to base 246 of SEQ ID NO:2. Enhancer region 3 is located from base 246 to base 253 of SEQ ID NO:2 and has the nucleotide sequence TGACGTAA.

[0075] Another preferred embodiment of the invention is the DNA shown in SEQ ID NO:3, called the CmpS promoter. The CmpS promoter also is a fragment of the sequence shown in SEQ ID NO: 1 and contains the 346 bp of SEQ ID NO:2 plus 54 bp from the leader region of the CmYLCV full-length transcript promoter. The leader region is a nucleotide sequence preceding the coding region which is transcribed but not translated into protein. It is known to the skilled artisan that the leader region can contain regulatory elements with important functions in gene expression. The CmpS promoter is obtainable by PCR with genomic DNA from Cestrum yellow leaf curling virus or from Cestrum yellow leaf curling virus-infected Cestrum parqui plants using forward primer Cmp1 (SEQ ID NO:13) and reverse primer CmpS2 (SEQ ID NO:15). The CmpS promoter contains at least 2 putative enhancer regions in the leader region. Enhancer region 4 (GAGAGA) is located at base 354 to base 359 of SEQ ID NO:3 and enhancer region 5 (GAGAGAGA) is located at base 368 to base 375 of SEQ ID NO:3.

[0076] Yet another preferred embodiment of the invention is the sequences depicted in SEQ ID NO:4, called the CmpL promoter. As the preceding promoters, the CmpL promoter is a fragment of the sequence shown in SEQ ID NO: 1 and contains the 346 bp of SEQ ID NO:2 plus 288 bp of the CmYLCV full-length transcript 5′ non-translated leader sequence. The CmpL promoter is obtainable by PCR with genomic DNA from Cestrum yellow leaf curling virus or from Cestrum yellow leaf curling virus-infected Cestrum parqui plants using forward primer Cmp1 (SEQ ID NO:13) and reverse primer CmpL2 (SEQ ID NO:16). A variant of SEQ ID NO:4, called CmpF, wherein the CmYLCV full-length transcript 5′ non-translated leader sequence is 286 bp instead of 288 bp in length, is shown in SEQ ID NO:20.

[0077] It is known to the skilled artisan that the nucleotide sequences shown in SEQ ID NOs:1, 2, 3, 4, 19 and 20 can be extended at their 5-′ and 3′-ends with homologous or heterologous nucleotide sequences. For example, the 5′-end of SEQ ID NOs:1, 2, 3, 4, 19 and 20 can be extended by all or part of the nucleotides shown in SEQ ID NO:6. SEQ ID NO:6 is naturally found upstream of SEQ ID NOs: 2, 3, 4 and 20 and contains part of ORF VI of CmYLCV (base 1 to base 100 of SEQ ID NO:6) as well as part of the CmYLCV full-length transcript promoter (base 101 to base 104 of SEQ ID NO:6). Likewise, the 3′-ends of SEQ ID NOs:2, 3, 4 and 20 can be extended by all or part of the nucleotides shown in SEQ ID NO:5, representing the nucleotide sequence naturally found at the 3′-end of SEQ ID NOs:4 and 20 and comprising part of the CmYLCV full-length transcript leader sequence.

[0078] As described above, the DNA sequences of the invention can be obtained, for example, by PCR with genomic DNA from Cestrum yellow leaf curling virus or from Cestrum yellow leaf curling virus-infected Cestrum parqui plants. Alternatively, the DNA sequences of the invention can be obtained from any other virus of the Caulimovirus family comprising homologues of the DNA sequence of the invention using sequence specific primers.

[0079] It is apparent to the skilled artisan that, based on the nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:6 and SEQ ID Nos 19 and 20, any primer combination of interest can be chosen to PCR amplify DNA fragments of various lengths that can be used according to the invention. The invention thus includes fragments derived from the CmYLCV full-length transcript promoters that function according to the invention, i.e. are capable of conferring expression of an associated nucleotide sequence.

[0080] This can be tested by generating such promoter fragments, fusing them to a selectable or screenable marker gene and assaying the fusion constructs for retention of promoter activity. Such assays are within the ordinary skill of the person skilled in the art. Preferred DNA fragments of the invention are of at least about 50 bases, preferably of between about 400 bases and about 650 bases, more preferably of between about 200 bases and about 400 bases and most preferably of about 350 bases in length.

[0081] It is also clear to the skilled artisan that mutations, insertions, deletions and/or substitutions of one or more nucleotides can be introduced into the DNA sequences of SEQ ID NOs:1, 2, 3, 4, 5 and 6 or longer or shorter fragments derived from the sequence information thereof using methods known in the art. In addition, an unmodified or modified nucleotide sequence of the present invention can be varied by shuffling the sequence of the invention. To test for a function of variant DNA sequences according to the invention, the sequence of interest is operably linked to a selectable or screenable marker gene and expression of the marker gene is tested in transient expression assays with protoplasts or in stably transformed plants. It is known to the skilled artisan that DNA sequences capable of driving expression of an associated nucleotide sequence are build in a modular way. Accordingly, expression levels from shorter DNA fragments may be different than the one from the longest fragment and may be different from each other. For example, deletion of a down-regulating upstream element will lead to an increase in the expression levels of the associated nucleotide sequence while deletion of an up-regulating element will decrease the expression levels of the associated nucleotide sequence. It is also known to the skilled artisan that deletion of development-specific or a tissue-specific element will lead to a temporally or spatially altered expression profile of the associated nucleotide sequence.

[0082] Embraced by the present invention are also functional equivalents of the promoters of the present invention, i.e. nucleotide sequences that hybridize under stringent conditions to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, such as, for example, the sequences shown in SEQ ID NO:19 and SEQ ID NO:20. A stringent hybridization is performed at a temperature of 65° C., preferably 60° C. and most preferably 55° C. in double strength (2×) citrate buffered saline (SSC) containing 0.1% SDS followed by rinsing of the support at the same temperature but with a buffer having a reduced SSC concentration. Such reduced concentration buffers are typically one tenth strength SSC (0.1×SSC) containing 0.1% SDS, preferably 0.2×SSC containing 0.1% SSC and most preferably half strength SSC (0.5×SSC) containing 0.1% SDS. In fact, functional equivalents to all or part of the CmYLCV full-length transcript promoter from other organisms can be found by hybridizing any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:19 or SEQ ID NO:20 with genomic DNA isolated from an organism of interest, particularly from another Caulimovirus. The skilled artisan knows how to proceed to find such sequences as there are many ways known in the art to identify homologous sequences in other organisms. Such newly identified DNA molecules then can be sequenced and the sequence can be compared to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:19 or SEQ ID NO:20 and tested for promoter activity. Within the scope of the present invention are DNA molecules having at least 75%, preferably 80%, more preferably 90%, and most preferably 95% sequence identity to the nucleotide sequence of any one of SEQ ID Nos:1, 2, 3, 4 or 6 over a length of at least 50 nucleotides. The percentage of sequence identity is determined using computer programs that are based on dynamic programming algorithms. Computer programs that are preferred within the scope of the present invention include the BLAST (Basic Local Alignment Search Tool) search programs designed to explore all of the available sequence databases regardless of whether the query is protein or DNA. Version BLAST 2.0 (Gapped BLAST) of this search tool has been made publicly available on the Internet (currently http://www.ncbi.nlm.nih.gov/BLAST/). It uses a heuristic algorithm which seeks local as opposed to global alignments and is therefore able to detect relationships among sequences which share only isolated regions. The scores assigned in a BLAST search have a well-defined statistical interpretation. Said programs are preferably run with optional parameters set to the default values.

[0083] It is another object of the present invention to provide recombinant DNA molecules comprising a DNA sequence according to the invention operably linked to a nucleotide sequence of interest. The nucleotide sequence of interest can, for example, code for a ribosomal RNA, an antisense RNA or any other type of RNA that is not translated into protein. In another preferred embodiment of the invention the nucleotide sequence of interest is translated into a protein product. The nucleotide sequence associated with the promoter sequence may be of homologous or heterologous origin with respect to the plant to be transformed. The sequence may also be entirely or partially synthetic. Regardless of the origin, the associated DNA sequence will be expressed in the transformed plant in accordance with the expression properties of the promoter to which it is linked. In case of homologous nucleotide sequences associated with the promoter sequence, the promoter according to the invention is useful for ectopic expression of said homologous sequences. Ectopic expression means that the nucleotide sequence associated with the promoter sequence is expressed in tissues and organs and/or at times where said sequence may not be expressed under control of its own promoter. In one particular embodiment of the invention, expression of nucleotide sequence associated with the promoter sequence suppresses its own expression and that of the original copy of the gene by a process called cosuppression.

[0084] In a preferred embodiment of the invention the associated nucleotide sequence may code for a protein that is desired to be expressed throughout the plant at all times and in most tissues and organs. Such nucleotide sequences preferably encode proteins conferring a desirable phenotypic trait to the plant transformed therewith. Examples are nucleotide sequences encoding proteins conferring antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, improved nutritional value, improved performance in an industrial process or altered reproductive capability. The associated nucleotide sequence may also be one that is transferred to plants for the production of commercially valuable enzymes or metabolites in the plant. Embraced by the present invention are also selectable or screenable marker genes, i.e. genes comprising a DNA sequence of the invention operably linked to a coding region encoding a selectable or screenable trait.

[0085] Examples of selectable or screenable marker genes are described below. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptII gene which confers resistance to kanamycin, paromomycin, geneticin and related antibiotics (Vieira and Messing, 1982, Gene 19: 259-268; Bevan et al., 1983, Nature 304:184-187) the bacterial aadA gene (Goldschmidt-Clermont, 1991, Nucl. Acids Res. 19: 4083-4089), encoding aminoglycoside 3′-adenylyltransferase and conferring resistance to streptomycin or spectinomycin, the hph gene which confers resistance to the antibiotic hygromycin (Blochlinger and Diggelmann, 1984, Mol. Cell. Biol. 4: 2929-2931), and the dhfr gene, which confers resistance to methotrexate (Bourouis and Jarry, 1983, EMBO J. 2: 1099-1104). Other markers to be used include a phosphinothricin acetyltransferase gene, which confers resistance to the herbicide phosphinothricin (White et al., 1990, Nucl. Acids Res. 18: 1062; Spencer et al. 1990, Theor. Appl. Genet. 79: 625-631), a mutant EPSP synthase gene encoding glyphosate resistance (Hinchee et al., 1988, Bio/Technology 6: 915-922), a mutant acetolactate synthase (ALS) gene which confers imidazolione or sulfonylurea resistance (Lee et al., 1988, EMBO J. 7: 1241-1248), a mutant psbA gene conferring resistance to atrazine (Smeda et al., 1993, Plant Physiol. 103: 911-917), or a mutant protoporphyrinogen oxidase gene as described in EP 0 769 059. Identification of transformed cells may also be accomplished through expression of screenable marker genes such as genes coding for chloramphenicol acetyl transferase (CAT), β-glucuronidase (GUS), luciferase (LUC), and green fluorescent protein (GFP) or any other protein that confers a phenotypically distinct trait to the transformed cell. Selection markers resulting in positive selection, such as a phosphomannose isomerase gene, as described in patent application WO 93/05163, are also used. Other genes to be used for positive selection are described in WO 94/20627 and encode xyloisomerases and phosphomanno-isomerases such as mannose-6-phosphate isomerase and mannose-1-phosphate isomerase; phosphomanno mutase; mannose epimerases such as those which convert carbohydrates to mannose or mannose to carbohydrates such as glucose or galactose; phosphatases such as mannose or xylose phosphatase, mannose-6-phosphatase and mannose-1-phosphatase, and permeases which are involved in the transport of mannose, or a derivative, or a precursor thereof into the cell. The agent which reduces the toxicity of the compound to the cells is typically a glucose derivative such as methyl-3-O-glucose or phloridzin. Transformed cells are identified without damaging or killing the non-transformed cells in the population and without co-introduction of antibiotic or herbicide resistance genes. As described in WO 93/05163, in addition to the fact that the need for antibiotic or herbicide resistance genes is eliminated, it has been shown that the positive selection method is often far more efficient than traditional negative selection.

[0086] Therefore, the promoters of the present invention are preferably operably linked to a nucleotide sequence which encodes a protein which comprises a region which: (a) encodes a protein which is involved in the metabolism of the compound, and/or (b) regulates the activity of the gene encoding the protein, wherein the compound is mannose or xylose or a derivative or a precursor of these, or a substrate of the protein, or is capable of being metabolized by the transformed cells into such a substrate. Said nucleotide sequence may encode a mannose-6-phosphate isomerase and the compound may be mannose. Alternatively, the nucleotide sequence may encode a phosphosugar-isomerase, a phosphosugar-mutase such as phosphomanno mutase, a phosphatase such as mannose-6-phosphatase, a sugar-epimerase, a sugar-permease, a phosphosugar-permease or a xylose isomerase, and the compound may be mannose, mannose-6-phosphate, D-mannoseamine or xylose.

[0087] In a preferred embodiment of the invention, the promoters of the invention are operably linked to the coding region of the E. coli manA gene (Joersbo and Okkels, 1996, Plant Cell Reports 16:219-221, Negrotto et al., 2000, Plant Cell Reports 19:798-803), encoding a phosphomannose isomerase (PMI), and a Nos (nopaline synthetase) terminator obtained from Agrobacterium tumefaciens T-DNA (Depicker et al., J. Mol. Appl. Genet. 1 (6), 561-573 (1982)). The skilled artisan knows that the Nos terminator may be substituted for by any other terminator that functions in plants. The promoter of the invention may be the CmpD, CmpC, CmpS, CmpL, CmpB, CmpA, CmpE or CmpF promoter, depicted in SEQ ID Nos 1 to 6 and 19 to 20, or any promoter derived therefrom. In an even more preferred embodiment of the invention, the promoter is the CmpS promoter depicted in SEQ ID NO:3.

[0088] The promoters of the present invention may also be used to provide resistance or tolerance to viruses by operatively linking the promoters of the invention with coding regions of genes from viruses to be controlled.

[0089] For virus control of negative strand viruses such as tomato spotted wilt virus (TSWV), the viral nucleocapsid protein (NP) and the movement protein (MP) gene sequences can be used, and for viruses belonging to the Alpha-like supergroup of viruses such as TMV and CMV, RNA-dependent RNA-polymerase (RdRpu) and movement protein (MP) gene sequences can be used. For viruses belonging to the Picoma-like supergroup of viruses, including potyviruses, any viral sequence may be linked to the promoters of the invention. Finally, for all other viruses, including DNA viruses, RNA-dependent RNA-polymerase (RdRp) or replicase-associated gene sequences may be used. Such constructs for virus control can be constructed as exemplified in Example 8 and Example 9 of WO 00/68374. It is within the ordinary skill of the person skilled in the art to replace the promoters disclosed in WO 00/68374 with the promoters of the present invention. Following the teaching of WO 00/68374 the skilled artisan also knows how to prepare constructs comprising the promoters of the invention operably linked to a viral sequences as mentioned hereinbefore to confer resistance or tolerance to said virus.

[0090] Instead of expressing double stranded viral RNA in transgenic plants, as disclosed in WO 00/68374, the viral RNA can also be expressed as a non-translatable RNA plus-sense viral RNA molecule as described, for example, in U.S. Pat. No. 558,302 or in Examples 12 and 13 of the present invention.

[0091] It is a further objective of the invention to provide recombinant expression vectors comprising a DNA sequence of the invention fused to an associated nucleotide sequence of interest. In these vectors, foreign DNA can be inserted into a polylinker region such that these exogenous sequences can be expressed in a suited host cell which may be, for example, of bacterial or plant origin. For example, the plasmid pBI101 derived from the Agrobacterium tumefaciens binary vector pBIN19 allows cloning and testing of promoters using beta-glucuronidase (GUS) expression signal (Jefferson et al, 1987, EMBO J. 6: 3901-3907). The size of the vector is 12.2 kb. It has a low-copy RK2 origin of replication and confers kanamycine resistance in both bacteria and plants. There are numerous other expression vectors known to the person skilled in the art that can be used according to the invention.

[0092] It is a further objective of the present invention to provide transgenic plants comprising the recombinant DNA sequences of the invention. The invention thus relates to plant cells, to plants derived from such cells, to plant material, to the progeny and to seeds derived from such plants, and to agricultural products with improved properties obtained by any one of the transformation methods described below. Plants transformed in accordance with the present invention may be monocots or dicots and include, but are not limited to, rice, maize, wheat, barley, rye, sweet potato, sweet corn, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar-beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, potato, eggplant, cucumber, Arabidopsis thaliana, and woody plants such as coniferous and deciduous trees. Preferred plants to be transformed are rice, maize, wheat, barley, cabbage, cauliflower, pepper, squash, melon, soybean, tomato, sugar-beet, sunflower or cotton, but especially rice, maize, wheat, Sorghum bicolor, orchardgrass, sugar beet and soybean. The recombinant DNA sequences of the invention can be introduced into the plant cell by a number of well-known methods. Those skilled in the art will appreciate that the choice of such method might depend on the type of plant which is targeted for transformation, i.e., monocot or dicot. Suitable methods of transforming plant cells include microinjection (Crossway et al., 1986, Bio Techniques 4:320-334), electroporation (Riggs and Bates, 1986, Proc. Natl. Acad. Sci., USA 83:5602-5606), Agrobacterium-mediated transformation (Hinchee et al., 1988, Bio/Technology 6:915-922; EP 0 853 675), direct gene transfer (Paszkowski et al., 1984, EMBO J. 3:2717-2722), and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del. (see, for example, U.S. Pat. No. 4,945,050 and McCabe et al., 1988, Bio/Technology 6:923-926). The cells to be transformed may be differentiated leaf cells, embryogenic cells, or any other type of cell. In the direct transformation of protoplasts, the uptake of exogenous genetic material into a protoplast may be enhanced by the use of a chemical agent or an electric field. The exogenous material may then be integrated into the nuclear genome. The previous work is conducted in dicot tobacco plants, which resulted in the foreign DNA being incorporated and transferred to progeny plants (Paszkowski et al., 1984, EMBO J. 3:2712-2722; Potrykus et al., 1985, Mol. Gen. Genet 199:169-177). Monocot protoplasts, for example, of Triticum monococcum, Lolium multiflorum (Italian rye grass), maize, and Black Mexican sweet corn, are transformed by this procedure. An additional preferred embodiment is the protoplast transformation method for maize as disclosed in EP 0 292 435, as well as in EP 0 846 771. For maize transformation also see Koziel et al., 1993, Bio/Technology 11:194-200. Transformation of rice can be carried out by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protoplast-mediated transformation is described for Japonica-types and Indica-types (Zhang et al., 1988, Plant Cell Rep., 7:379-384; Shimamoto et al., 1989, Nature 338:274-276; Datta et al., 1990, Bio/Technology 8:736-740). Both types described above are also routinely transformable using particle bombardment (Christou et al., 1991, Bio/Technology 9:957-962). Patent application No. EP 0 332 581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of all Pooideae plants including Dactylis and wheat. Furthermore, wheat transformation is described in patent application No. EP 0 674 715; and by Weeks et al., 1993 (Plant Physiol. 102:1077-1084). The thus-constructed plant expression vector can, for example, be introduced into the calli of rice according to the conventional plant transformation method, and the differentiation of roots and leaves is induced therefrom, and then, can be transferred to a flowerpot for cultivation, thereby obtaining the transformed rice.

[0093] The plants resulting from transformation with the DNA sequences or vectors of the present invention will express a nucleotide sequence of interest throughout the plant and in most tissues and organs.

[0094] The genetic properties engineered into the transgenic plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants. Generally said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting. Specialized processes such as hydroponics or greenhouse technologies can also be applied. Use of the advantageous genetic properties of the transgenic plants according to the invention can further be made in plant breeding that aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering. The various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants. Depending on the desired properties different breeding measures are taken. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines. Thus, the transgenic plants according to the invention can be used for the breeding of improved plant lines that for example increase the effectiveness of conventional methods such as herbicide or pesticide treatment or allow to dispense with said methods due to their modified genetic properties. Alternatively new crops with improved stress tolerance can be obtained that, due to their optimized genetic “equipment”, yield harvested product of better quality than products that were not able to tolerate comparable adverse developmental conditions.

[0095] It is another objective of the present invention to provide DNA sequences that can be used to express a nucleotide sequence of interest in a desired organism. This organism can be a bacterium, a plant or any other organism of interest.

[0096] Furthermore, the disclosure of SEQ ID NOs:1 to 6 enables a person skilled in the art to design oligonucleotides for polymerase chain reactions which attempt to amplify DNA fragments from templates comprising a sequence of nucleotides characterized by any continuous sequence of 15 and preferably 20 to 30 or more base pairs in SEQ ID Nos:1, 2, 3, 4, 5 or 6. Said nucleotides comprise a sequence of nucleotides which represents 15 and preferably 20 to 30 or more base pairs of SEQ ID Nos:1, 2, 3, 4, 5 or 6. Polymerase chain reactions performed using at least one such oligonucleotide and their amplification products constitute another embodiment of the present invention.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

[0097] SEQ ID NO:1 CmpD

[0098] SEQ ID NO:2 CmpC

[0099] SEQ ID NO:3 CmpS

[0100] SEQ ID NO:4 CmpL

[0101] SEQ ID NO:5 CmpB

[0102] SEQ ID NO:6 CmpA

[0103] SEQ ID NO:7 S1 forward primer

[0104] SEQ ID NO:8 S2 reverse primer

[0105] SEQ ID NO:9 GUS forward primer

[0106] SEQ ID NO:10 GUS reverse primer

[0107] SEQ ID NO:11 CAT forward primer

[0108] SEQ ID NO:12 CAT reverse primer

[0109] SEQ ID NO:13 Cmp1 forward primer

[0110] SEQ ID NO:14 CmpC2 reverse primer

[0111] SEQ ID NO:15 CmpS2 reverse primer

[0112] SEQ ID NO:16 CmpL2 reverse primer

[0113] SEQ ID NO:17 PA forward primer

[0114] SEQ ID NO:18 PA reverse primer

[0115] SEQ ID NO:19 CmpE

[0116] SEQ ID NO:20 CmpF

[0117] SEQ ID NO:21 NOS terminator forward primer

[0118] SEQ ID NO:22 NOS terminator reverse primer

[0119] SEQ ID NO:23 TSWV NP gene forward primer

[0120] SEQ ID NO:24 TSWV NP gene reverse primer

[0121] SEQ ID NO:25 CmYLCV promoter forward primer

[0122] SEQ ID NO:26 CmYLCV promoter reverse primer

[0123] SEQ ID NO:27 AGLINK multicloning site

[0124] SEQ ID NO:28 BIGLINK multicloning site

[0125] SEQ ID NO:29 Cmpf-B primer

[0126] SEQ ID NO:30 CmpS-B primer

[0127] SEQ ID NO:31 pNOV2804; binary vector, contains a promoterless PMI-nos terminator expression cassette

[0128] SEQ ID NO:32 pNOV3604; vector for transformation by biolistics, contains a promoterless PMI-nos terminator expression cassette

[0129] SEQ ID NO:33 CmpMr reverse primer

[0130] SEQ ID NO:34 CmpMf forward primer

[0131] SEQ ID NO:35 CmpMrs reverse primer

[0132] SEQ ID NO:36 CmpMfs forward primer

[0133] SEQ ID NO:37 synGFPI; plant optimized GFP gene with intron

[0134] SEQ ID NO:38 GIG; GUS gene with intron

[0135] SEQ ID NO:39 Cmp-C reverse primer

[0136] SEQ ID NO:40 CmpS-synGFPI-nos expression cassette

[0137] SEQ ID NO:41 CmpS-GIG-nos expression cassette

[0138] SEQ ID NO:42 CmpC-synGFPI-nos expression cassette

[0139] SEQ ID NO:43 CmpC-GIG-nos expression cassette

[0140] SEQ ID NO:44 pNOV2117 vector

[0141] SEQ ID NO:45 pNOV4200 vector

[0142] SEQ ID NO:46 ZmUbi-GFP-35S term expression cassette

[0143] SEQ ID NO:47 ZmUbi-GIG-nos expression cassette

[0144] SEQ ID NO:48 Ubq3(At)-synGFPI-nos expression cassette

[0145] SEQ ID NO:49 Ubq3(At)-GIG-nos expression cassette

EXAMPLES

[0146] Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described, for example, by Sambrook et al., 1989, “Molecular Cloning”, Cold Spring Harbor, Cold Spring Harbor Laboratory Press, NY and by Ausubel et al., 1994, “Current protocols in molecular biology”, John Wiley and Sons, New York.

Example 1 Virus Cloning

[0147] 1. DNA extraction Virus genomic DNA is extracted from Cestrum yellow leaf curling virus-infected Cestrum parqui plants. Five grams of infected leaves are homogenized in 30 ml of grinding buffer (0.2 M Tris pH 7.0, 0.02 M EDTA, 2 M Urea) for 60 sec at maximum speed in a Brinkman Polytron homogenizer with PT10 probe and gently shaken at 4° C. overnight with Triton X-100 (2% final concentration). Virus is purified from crude homogenate by low-speed centrifugation (10,000 rpm for 20 min in a Sorvall SS-34 rotor) and from the obtained supernatant by high-speed centrifugation (27,000 rpm for 2 hr in a Beckman SW-28 rotor) through a sucrose cushion (3 ml of 15% sucrose). The pellet-containing virus is then resuspended in 0.1 M Tris, pH 7.4, 2.5 mM MgCl₂. DNase I (Sigma) and RNase A (Sigma) are added to a concentration of 10 mg/ml each. After 30 min at 37° C., the reaction is stopped with the addition of EDTA to 10 mM. Virus DNA is isolated from CmYLCV particles by treating with protease K (E. Merck, 0.5 mg/ml final concentration) in the presence of 1% SDS at 65° C. for 15 min. The viral DNA is then purified by phenol extraction and by ethanol precipitation. The virus DNA contained in the final ethanol precipitate is dissolved in water.

[0148] 2. DNA amplification and cloning Hundred nanograms of the obtained DNA are used as a template for PCR amplification in a 50 μl reaction volume containing 10 μM of each primer, 25 mM each dNTP, 5 μl of pfu reaction buffer (Stratagene) and 2.5 units of pfu turbo DNA polymerase (Stratagene). A S1 forward primer (gaccacaaacatcagaag, SEQ ID NO:7) and a S2 reverse primer (caaacttattgggtaatc, SEQ ID NO:8) are used for the PCR reaction. Cycling parameters for PCR are: 1×(94° C. for 1 min); 30×(94° C. for 1 min, 50° C. for 1 min, 72° C. for 1 min); 1×(72° C. for 10 min) Each single DNA fragment is cloned in pPCR-Script™ Amp SK(+) plasmid (Stratagene) according to the manufacturer's instructions.

Example 2 DNA Sequencing

[0149] Sequencing of the DNA virus clones is carried out using the automated ABI PRISM 377 DNA sequencer (Perkin Elmer) and ABI PRISM dRhodamine terminator cycle sequencing kit (Perkin Elmer) according to the manufacturer's instructions. The described S1 primer and S2 primers (Example 1) are used for the sequencing reactions as well as the universal M13-20 and Reverse primers.

Example 3 Construction of Plasmids for Transient Expression

[0150] All the PCR reactions are carried out with Pfu polymerase (Stratagene) as described in Example 1.

[0151] 1. Reporter genes amplification The beta-glucuronidase (GUS) and the chloramphenicol acetyltransferase (CAT) reporter genes are amplified. For GUS gene amplification GUS forward (cagggtaccactaaaatcacc, SEQ ID NO:9) and GUS reverse (aggggatccccaattcccc, SEQ ID NO:10) oligonucleotides are used at the annealing temperature of 60° C. CAT forward (aggggtaccatcgatatggag, SEQ ID NO:11) and Cat reverse (ttaggatccgccctgccac, SEQ ID NO:12) oligonucleotides are annealed at 62° C. The forward primers are designed to contain a KpnI recognition site and the reverse ones a BamHI site.

[0152] 2. CmYLCV promoter amplification Three different promoter fragments are selected. CmpC (SEQ ID NO:2), CmpS (SEQ ID NO:3), and CmpL (SEQ ID NO:4). Cmp1 forward primer (cttctagacaaagtggcagac, SEQ ID NO:13) is used for all the three amplifications at the annealing temperature of 52° C. It is modified (shown in bold) from the original sequence to contain a XbaI recognition site. The CmpC2 reverse primer (ttggtaccttaacaatgaggg, SEQ ID NO:14) is used for CmpC fragment amplification and the CmpS2 reverse primer (ctacttctaggtaccttgctc, SEQ ID NO:15) is used for the CmpS fragment. Both of them are modified to contain a KpnI recognition site. The CmpL2 reverse primer (ttggtaccttaacaatgaggg, SEQ ID NO:16) is used for CmpL fragment amplification and it is modified to contain a ClaI restriction site.

[0153] 3. Polyadenylation signal amplification The polyadenylation signal fragments is amplified from the cauliflower mosaic virus (CaMV) infectious clone (Franck et al., Cell 21 (1), 285-294, 1980). PA (polyadenylation signal amplification) forward (ggggatccccagtctctctc, SEQ ID NO:17) and PA reverse (gtgaattcgagctcggta, SEQ ID NO:18) oligonucleotides are used for PCR and annealed at 60° C. The forward primer is designed to contain a BamHI recognition site and the reverse one an EcoRI site.

[0154] 4. Produced Constructs

[0155] pCmpCG (CmpC promoter fragment+GUS gene+polyA)

[0156] pCmpSG (CmpS promoter fragment+GUS gene+polyA)

[0157] pCmpCC (CmpC promoter fragment+CAT gene+polyA)

[0158] pCmpSC (CmpS promoter fragment+CAT gene+polyA)

[0159] pCmpLC (CmpL promoter fragment+CAT gene+polyA)

[0160] The cassettes containing the promoter fragment, the reporter genes and the polyadenylation signal are inserted in a pUC19 vector (Stratagene) restricted with XbaI and EcoRI.

[0161] 5. Constructs Used for Comparison

[0162] pCapG (Cap promoter fragment+GUS gene+polyA)

[0163] pCapSG (CapS promoter fragment+GUS gene+polyA)

[0164] pCapC (Cap promoter fragment+CAT gene+polyA)

[0165] Promoter fragments contained in these constructs are obtained from CaMV (Franck et al., Cell 21 (1), 285-294, 1980, see GenBank accession number V00141). GUS, CAT and polyA fragments are identical to those used for the CmYLCV constructs. Cap corresponds to the fragment from base −227 to base +33 from TATA-box (base 7175 to base 7438 of the CaMV genome, see GenBank accession number V00141) and CapS corresponds to the fragment from base −227 to base +82 from TATA-box (base 7175 to base 7486 of the CaMV genome, see GenBank accession number V00141). These positions correspond approximately to those chosen for the CmYLCV fragments.

Example 4 Transient Expression Experiments

[0166] 1. Suspension Cultures and Protoplast Preparation

[0167]Orychophragmus violaceus. Suspension cultures are maintained in 40 ml of MS medium (Murashige and Skoog, Physiol Plant 15, 474-497, 1962) including 100 mg/ml inositol, 2% sucrose, and 0.1 mg/ml 2,4D. Protoplasts are isolated from 4- to 5-day-old-cultures. Cell walls are digested at 26° C. for 1 hr in 0.1% pectolyaseu Y23 (Seishin Pharmaceutical Co., Japan), 1% cellulase Onozuka R10 (Yakult Honsha Co., Japan), 0.4 M D-mannitol, and 0.1% MES, pH 5.5. Protoplasts are filtered through a 50 μm sieve and washed twice with electroporation (EP) solution (10 mM HEPES, 150 mM NaCl, 5 mM CaCl₂, 0.2 M mannitol, pH 7.1).

[0168]Nicotiana plumbaginifolia. Plants are maintained axenically on RPM2 medium (Blonstein et al., Mol Gen Genet 211, 252-259, 1988) plus 7 g/l bacto agar, pH 5.6. For protoplasts preparation, leaves are cut and incubated overnight at 26° C. in a solution of 0.5% driselase (Fluka), 0.25 mM PVP 10 (polyvinylpyrrolidone MW 10000), 3.85 mM CaCl₂, 6 mg/l NAA, 2 mg/l BAP, and 0.5 M sucrose, pH 5.7. Protoplasts are filtered through a 100 μm sieve. Sucrose solution (0.6 M sucrose, 0.1% MES, and 15 mM CaCl₂, pH 5.7) is added to the protoplast suspension for the first purification step, and the suspension is overlaid with W5 solution (150 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 6 mM glucose; Menczel et al., Theor Appl Genet 59, 191-195, 1981). Protoplasts are then washed once with W5 solution and finally with EP solution.

[0169]Oryza sativa. Protoplasts are prepared from a morphogenic rice suspension culture established from O. sativa cv. Nipponbare as described (Datta et al., 1990, Bio/Technology 8:736-740).

[0170] 2. Transient expression experiment Transfection by electroporation of 2×10⁶ Orychophragmus violaceus protoplasts in 0.66 ml EP buffer is carried out by discharging a 960 μF capacitor through a distance of 4 mm of protoplast suspension. The capacitor is loaded at the 450 Volts. Electroporation is performed in the presence of 5-10 μg of plasmid DNA, then protoplasts are cultivated 16 to 24 hours at 25° C. Transfection of 2×10⁶ Nicotiana plumbaginifolia protoplast in 0.3 ml suspension is carried out in the presence of 0.3 ml PEG (40% polyetyleneglycole 6000) and 5-10 μg of plasmid DNA. Protoplasts are cultivated in 0.4 ml K3 medium (Godall et al., Methods Enzymol 181, 148-161, 1990) for 16 to 24 hours at 25° C. and added with 10 ml W5 buffer before harvesting.

[0171] Protein extracts are prepared by at least three cycles of freezing and thawing, and clarified by centrifugation.

Example 5 CAT and GUS Assays

[0172] For detection of CAT gene expression double antibody sandwich (DAS)-ELISA is carried out using a CAT ELISA kit (Boehringer) according to the manufacturer's instructions. CAT activity is measured with a Dynex MRXII apparatus. Samples for GUS assay are diluted in GUS buffer (0.05 M NaPO₄, pH 7, 0.01 M EDTA, 0.1% Triton-X-100, 0.1% Sarkosyl). The reaction is carried out in the presence of an equal amount of GUS-reaction buffer (100 ml/l 10×GUS buffer, 200 mg/l BSA, 705 mg/l 4-methylumbelliferyl-glucuronide) at 37° C. and stopped with 2 M Ammediol. The activity is measured in a Titertek Fluoroskan II.

[0173] Both CAT and GUS assay results are normalized to a standard clone value. Results obtained from ten different experiments show that the various CmYLCV promoter fragments function as highly active promoters.

[0174]N. plumbaginifolia Protoplast Transient Expression GUS reporter gene CmpCG 100 CmpSG 86.9 +/− 20% CapG   9 +/− 20% CapSG 38.9 +/− 15% CAT reporter gene CmpCC 100 CmpSC   78 /− 20% CapSC 89.5 +/− 15% CmpLC   9 +/− 20%

[0175]O. violaceus Protoplast Transient Expression GUS reporter gene CmpCG 100 CmpSG 84.6 +/− 15% CapG  9.6 +/− 15% CapSG 40.7 +/− 15% CAT reporter gene CmpCC 100 CmpSC  255 +/− 20% CapSC  546 +/− 15% CmpLC   10 +/− 20%

[0176]O. sativa Protoplast Transient Expression GUS reporter gene CmpCG 100 CmpSG 84.6 +/− 15% CapG  9.6 +/− 15% CapSG 40.7 +/− 15%

Example 6 Preparation of Solutions and Media for Plant Regeneration and Transformation

[0177] Culture media GM, CIM and SIM are the media described by Valvekens et al. (1988, Proc. Natl. Acad. Sci. USA. 85: 5536-5540).

[0178] Culture medium GM contains the mineral salts of Murashige and Skoog (1962, Physiol. Plant. 15:473-497), 1.0 mg/l thiamine (stock 1 mg/ml), 0.5 mg/l pyridoxine HCl (stock 1 mg/ml), 0.5 mg/[nicotinic acid (stock 1 mg/ml), 0.5 g/l 2-(N-morpholino)ethanesulfonic acid (MES), 10 g/l sucrose, 8 g/l agar, with the pH adjusted to 5.8 with 1 N KOH. CIM contains the mineral salts and vitamins of B5 medium (Gamborg et al., 1968, Exp. Cell Res. 50:151-158), 0.5 g/l 2-(N-morpholino)ethanesulfonic acid (MES), 20 g/l glucose, 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) (stock 10 mg/ml in DMSO), 0.05 mg/l kinetin (stock 5 mg/ml in DMSO), pH 5.8. Solid CIM medium contains 8 g/l agar. SIM contains the mineral salts and vitamins of B5 medium (Gamborg et al., 1968, supra), 0.5 g/l 2-(N-morpholino)ethanesulfonic acid (MES), 20 g/l glucose, 5 mg/l N6-(2-isopentenyl)adenine (2iP) (stock 20 mg/ml in DMSO), 0.15 mg/l indole-3-acetic-acid (IAA) (stock 1.5 mg/ml in DMSO), 8 g/l agar, pH 5.8. SIM V750 K100 is SIM medium supplemented with 750 mg/l vancomycin and 100 mg/l kanamycin. SIM V500 K100 is SIM medium supplemented with 500 mg/l vancomycin and 100 mg/l kanamycin. GM K50 is GM medium supplemented with 50 mg/l kanamycin.

[0179] The culture media are all sterilized by autoclaving (20 min, 121° C.). Vitamins are dissolved in water and added to media before autoclaving. Hormones are dissolved in dimethyl sulfoxide (DMSO). Antibiotics are dissolved in water and sterilized by filtration (0.22 μm). Hormones and antibiotics are added after autoclaving and cooling of the media to 65° C. In all cases 9-cm Petri dishes (Falcon, 3003) are used, except for GM and GM K50 which are usually poured into 15-cm Petri dishes (Falcon, 3025).

[0180] Plates with solid media are dried before usage in laminar flow to remove condensate.

Example 7 Arabidopsis strain and Growth Conditions

[0181]Arabidopsis thaliana seeds ecotype Columbia (Col-0) wild type are purchased from Lehle Seeds, USA (1102 South Industrial Blvd. Suite D, Round Rock Tex. 78681, USA). Plants are grown at 22° C. 16/8 hour light/dark cycle in pots in the mixture of 4 parts sand, 4 parts garden soil and 1 part agrilit.

Example 8 Agrobacterium Strain and Culture

[0182] Vector plasmids are introduced into recipient Agrobacterium tumefaciens strain LBA4404 (Clontech) by triparental mating according to the protocol described by Walkerpeach and Velten (“Agrobacterium-mediated gene transfer to plant cells: Cointegrate and binary vector systems”. in: Plant Molecular Biology Manual, B1: 1-19,1994. Eds.: S. B. Gelvin, R. A., Schilperoort, Kluvers Acad. Publishers.). Mobilizing strain used is E. coli HB101 harboring conjugation plasmid pRK2013 (Ditta et al., 1980, Broad host range DNA cloning system from Gram-negative bacteria. Construction of gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA 77: 7347-7351). Agrobacteria used for root transformation are grown in LB medium (Sambrook et al., 1989, “Molecular Cloning”, Cold Spring Harbor, Cold Spring Harbor Laboratory Press, NY) without antibiotics at 28° C. and 200 rpm.

Example 9 Seed Sterilization

[0183] Seeds are placed in 70% EtOH/0.05% Tween 20 for 1 minute in a 2 ml Eppendorf tube. 70% EtOH/0.05% Tween 20 is removed with a pipette and replaced with 5% NaOCl/0.05% Tween 20 for 15 minutes. Seeds are shaken regularly. The solution is removed in sterile conditions and the seeds are washed in sterile, distilled water 3 times for 10 minutes each. After the last wash seeds are keep in 0.5-1 ml water. Seeds can be used immediately or stored at 4° C. for two-three weeks. Sterilized seeds (20-30) are transferred with forceps on GM medium in 15-cm Petri dishes. Seedlings are grown in vertically placed plates in a growth chamber (22° C.; 16/8 hour light/dark cycle).

Example 10 Transformation of Root Explants of Arabidopsis thaliana

[0184] Roots of three-week-old seedlings are used in the transformation procedure. Roots should not be green or brown. Green parts of seedlings are removed with scalpel and forceps. Remaining roots are collected and approximately 5 entire root systems are placed per plate with solid CIM medium. Roots are pressed gently onto the surface of the plate to ensure full contact with the medium, but they should not be dipped into the agar. Roots are incubated for three days in a growth chamber (22° C.; 16/8 hour light/dark cycle). Roots are then transferred to a sterile Petri dish with filter paper wetted with liquid CIM medium and cut with a scalpel in 0.5-1 cm pieces. Root explants are then transferred to a 50 ml sterile Falcon tube containing 10 ml of liquid CIM medium. To this, 0.5 ml of an overnight Agrobacterium culture (OD 0.6-1) is added and incubate for 1-2 minutes while shaking gently. The liquid is poured out of the tube through sterile metal screens (50 mesh, Sigma, S-0895), which are kept with forceps. Roots usually remain on the wall of the tube close to its edge. Then the root explants are transferred to a sterile Petri dish with filter paper and briefly blotted dry to remove excess of liquid. Root explants are put onto plates with solid CIM medium and incubated in a growth chamber for 2 days under dim light (1.5-2 klux). Slight traces of overgrowth with Agrobacterium should be visible after the period of cocultivation. Root explants are then transferred to sterile 50 ml Falcon tubes with 20 ml of liquid CIM medium, supplemented with 1000 mg/l vancomycin. The Falcon tubes are then gently vortexed to remove the Agrobacteria. The liquid is poured out of the tube as described above and the explants are briefly blotted dry on filter paper. Explants are then transferred to plates containing SIM V750 K100 medium. Roots should be in a close contact with the medium. The explants are incubated in a growth chamber in normal conditions for one week and then transferred to SIM V500 K1100 medium and incubated for an additional week. Then the amount of vancomycin is reduced to 250 mg/l. First shoots should appear at the end of the third week of cultivation on SIM media. Shoots are excised when 0.3-0.5-cm long, any residual callus is removed, and the shoots are transferred to 15-cm plates containing GM K50 medium. Max. 3 shoots are placed per plate. To get more shoots, the remaining root explants can be transferred to fresh SIM plates supplemented with 125 mg/l vancomycin and 100 mg/l kanamycin for additional two weeks. Rooted shoots can be transferred to soil to allow seed set. Shoots that do not root are transferred to Magenta jars (one per jar) containing GM medium to produce seeds in vitro.

[0185] Seeds from individual transgenic plants are germinated on GM K50 medium in growth chamber for 2 weeks. Phenotypically normal kanamycin resistant seedlings, which form green true leaves and branched root system, are selected for further analyses.

Example 11 Histochemical β-glucuronidase (GUS) Assay

[0186] In vitro grown seedlings or plants grown in soil are used in GUS assays. Either whole seedlings or dissected organs are dipped into GUS staining solution. GUS staining solution contains 1 mM 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc, Duchefa, 20 mM stock in DMSO), 100 mM Na-phosphate buffer pH 7.0, 10 mM EDTA pH 8.0, and 0.1% Triton X100. Tissue samples are incubated at 37° C. for 1-16 hours. If necessary samples can be cleared with several washes of 70% EtOH to remove chlorophyll.

Example 12 Construction of Tobacco Transformation Vector pZU627

[0187] All the PCR reactions are carried out with Platinum Pfx DNA Polymerase (Life Technologies) according to manufacturer's instructions.

[0188] 1. NOS Terminator Amplification and Cloning

[0189] The NOS terminator is amplified from pBIN19 (Bevan et al 1984) using the NOS terminator forward primer (SEQ ID NO:21) and NOS terminator reverse primer (SEQ ID NO:22). The forward primer is modified to contain a PstI site and the reverse primer is modified to contain a HindIII site. The NOS terminator amplification product is cloned as PstI/HindIII fragment in same sites of pBluescript (Stratagene) resulting in plasmid pZU400A.

[0190] 2. Amplification of the Tomato Spotted Wilt Virus Nucleocapsid Protein (TSWV NP) Gene Amplification and Cloning

[0191] In order to obtain a non-translatable TSWV NP gene, modified TSWV NP gene primers are used to amplify the TSWV NP gene. The TSWV NP gene forward primer introduces a BamHI, a SphI, a start and a stop codon (SEQ ID NO:23). The reverse primer is modified to introduce a PstI site (SEQ ID NO:24). The amplification product is cloned as BamHI/PstI fragment into the BamHI/PstI site of pZU400A upstream of and in same direction as the NOS terminator. In the resulting clone pZU400b the PstI site between the TSWV NP gene and the NOS terminator is removed using a T4 DNA polymerase treatment (Life Technologies) according to manufacturer's instructions. From the resulting clone denoted pZU400C, the TSWV NP gene and NOS terminator are cloned as a SphI/HindIII fragment into the SphI/HindIII site of shuttle vector pZO1560 resulting in plasmid pZU400. pZO1560 is a pBluescript derivative in which the original multicloning site is replaced by the AGLINK multicloning site (SEQ ID NO:27).

[0192] 3. CmYLCV Promoter Amplification and Cloning

[0193] The CmYLCV viral promoter is amplified using CmYLCV promoter forward primer (SEQ ID NO:25) and CmYLCV promoter reverse primer (SEQ ID NO:26). The forward primer is modified to contain a SstI site and the reverse primer is modified to contain a PstI site. The amplified CmYLCV viral promoter is cloned as SstI/PstI fragment, in same direction as and upstream of the TSWV-N gene, into the SstI/PstI site of pZU400. This results in clone pZU625 that contains a viral gene cassette that produces a non-translatable TSWV NP RNA.

[0194] 4. Cloning of Viral Gene Cassette to pVictorHiNK

[0195] The viral gene cassette is cloned as AscI/PacI fragment from pZU625 into the AscI/PacI site of pVictorHiNK vector pZU494. This results in the plant transformation vector pZU627. pVictorHiNK is a binary vector comprising an origin of replication (ORI) derived from Pseudomonas aeruginosa plasmid pVS1 which is known to be highly stable in A. tumefaciens (Itoh et al., 1984. Plasmid 11:206-220; Itoh and Haas, 1985. Gene 36;27-36). The pVS1 ORI is only functional in Agrobacterium and can be mobilized by the helper plasmid pRK2013 from E. coli into A. tumefaciens by means of a triparental mating procedure (Ditta et al., 1980. Proc. Natl. Acad. Sci. USA 77:7347-7351).

[0196] This binary vector also comprises a ColEI origin of replication which is functional in E. coli and derived from pUC19 (Yannisch-Perron et al., 1985. Gene 33:103-119).

[0197] For maintenance in Ecoli and A. tumefaciens this vector contains a bacterial resistance gene to spectomycin and streptomycin encoded by a 0.93 kb gene from transposon Tn7 (Fling et al., 1985. Nucl. Acid Res. 13:7095) which functions as selection marker for maintenance of the vector in E. coli and A. tumefaciens. The gene is fused to the tac promoter for efficient bacterial expression (Amman et al., 1983. Gene 25: 167-178).

[0198] The right and left T-DNA border fragments of 1.9 kb and 0.9 kb, respectively that comprise the 24 bp border repeats, are derived from the Ti-plasmid of the nopaline type A. tumefaciens strains pTil37 (Yadev et al., 1982. Proc. Natl. Acad. Sci. USA. 79:6322-6326). Subsequently the T-DNA region between the borders is modified by deleting the M13 derived sequences and, to improve its cloning versatility, the BIGLINK multicloning site (SEQ ID NO:28) is introduced yielding pVictorHiNK.

[0199] PVictorHiNK contains an NPTII gene cassette for selection of transformants during the plant transformation process. This gene cassette contains a NOS promoter, the NPTII gene and the NOS terminator, obtained from A. tumefaciens.

Example 13 Plant Transformation and Screening for Resistance

[0200] 1. Transformation of Binary Vectors to Plant Material

[0201] Methods to transfer binary vectors to plant material are well established and known to a person skilled in the art. Variations in procedures exist due to for instance differences in used Agrobacterium strains, different sources of explant material, differences in regeneration systems depending on as well the cultivar as the plant species used. Binary vector pZU627 is used in plant transformation experiments according to the following procedures. PZU627 is transferred by tri-parental mating to an acceptor Agrobacterium tumefaciens strain, followed by Southern blot analysis of the ex-conjugants for verification of proper transfer of the construct to the acceptor strain, inoculation and co-cultivation of axenic explant material with the recombinant Agrobacterium tumefaciens strain, selective killing of the Agrobacterium tumefaciens strain using the appropriate antibiotics, selection of transformed cells by growing on selective media containing kanamycine, transfer of plantlets to soil, assaying for the intactness of the integrated T-DNA by Southern blot analysis of isolated chromosomal DNA of the plant.

[0202] 2. Resistance of Plants Against TSWV Infections

[0203] Transformed plants are grown in the greenhouse under standard quarantine conditions in order to prevent any infection by pathogens. At a four-leaf stage the plants are infected with TSWV by mechanical inoculation. Tissue from plants systemically infected with TSWV is ground in 5 volumes of ice-cold inoculation buffer (10 mM phosphate buffer supplemented with 1% Na₂SO₃) and rubbed in the presence of carborundum powder on the first two fully extended new leaves. The inoculated plants are monitored for symptom development during three weeks after inoculation. Plants containing pZU627 sequences do not show TSWV symptoms, whereas untransformed control plants show severe systemic TSWV symptoms within 7 days after inoculation. Resistant plants are self-pollinated and the seeds harvested. Progeny plants are analyzed for segregation of the inserted gene and subsequently re-screened for resistance against TSWV infection as described above.

Example 14 Construction of CmpS-Phosphomannose Isomerase-nos Constructs for Plant Transformation

[0204] The CmpS promoter is PCR amplified using the Cmpf-B (cgc gga tcc tgg cag aca aag tgg cag a; SEQ ID NO:29) and the CmpS-B (cgc gga tcc tac ttc tag gct act tg, SEQ ID NO:30) primers having flanking BamHI sites. The resulting PCR fragment is cloned into the BamHI site of pBluescript KS (+) to form pNOV4211.

[0205] To create a binary vector for transformation via Agrobacterium tumefaciens, the CmpS promoter is excised from pNOV4211 using BamHI and inserted into BamHI-digested pNOV2804 (SEQ ID NO:31) upstream of the PMI gene, and the resulting vector called pNOV2819. pNOV2804 (SEQ ID NO:31) is a binary vector with VS1 origin of replication, a copy of the Agrobacterium virG gene in the backbone, and a promoterless PMI-nos terminator expression cassette between the left and right borders of T-DNA. PMI (phosphomannose isomerase) is the coding region of the E. coli manA gene (Joersbo and Okkels, 1996, Plant Cell Reports 16:219-221, Negrotto et al., 2000, Plant Cell Reports 19:798-803). The nos (nopaline synthase) terminator is obtained from Agrobacterium tumefaciens T-DNA (Depicker et al., 1982, J. Mol. Appl. Genet. 1 (6), 561-573. The phosphomannose isomerase coding region and the nos terminator are located at nt 290 to nt 1465 and nt 1516 to 1789 respectively, of pNOV2804 (SEQ ID NO:31).

[0206] To create a vector for biolistic transformation, the CmpS promoter is excised from pNOV4211 using BamHI and inserted into BamHI-digested pNOV3604 (SEQ ID NO:32) to form pNOV2820, thus creating a PMI expression cassette driven by the CmpS promoter. pNOV3604 is a vector for preparing biolistic fragments and is based on pUC19 with an ColEI origin of replication and a beta-lactamase gene conferring ampicillin-resistance. As pNOV2804, pNOV3604 also contains a promoterless PMI-nos terminator expression cassette (see above). The phosphomannose isomerase coding region and the nos terminator are located at nt 42 to nt 1217 and nt 1268 to 1541 respectively, of pNOV3604 (SEQ ID NO:32).

[0207] Binary vector pNOV2819 is used for transformation via Agrobacterium tumefaciens and vector pNOV2820 is used for biolistic transformation.

Example 15 Construction of CmpC-GUS Plasmids for Stable Expression In Planta.

[0208] 1. Construction of the Binary Vector

[0209] The vector pCambia 1302 (Cambia, Canberra, Australia; Roberts, C. S., Rajagopal, S., Smith, L., Nguyen, T., Yang, W., Nugroho, S., Ravi, K. S. Cao, M. L., Vijayachandra, K., et al. A comprehensive set of modular vectors for advanced manipulation and efficient transformation of plants. Presented at the Rockefeller Foundation Meeting of the International Program on Rice Biotechnology, Malacca, Malaysia, Sep. 15-19, 1997) is chosen and modified for the experiments: The CaMV 35S promoter in front of the GFP gene is removed by digestion at position 9788 with HindIII endonuclease and at position 1 with NcoI endonuclease and the vector is relegated at the two compatible ends. Digestion with XhoI endonuclease at positions 7613 and BstXI at position 9494 excise the hygromycin gene and the CaMV 35S promoter in front of it. The 35S promoter sequences are eliminated from the vector to exclude any risk of homology-mediated gene silencing. The bar gene driven by the 1′ promoter (Mengiste et al., Plant J, 1997, 12(4): 945-948) is introduced in the EcoRI site at position 9737 as a selectable marker. The obtained vector is named pCamBar.

[0210] 2. Produced Constructs

[0211] The cassettes CmpCG and CapG described in Example 3 are inserted in the pCamBar vector between XbaI site at position 9764 and EcoRI at position 9737 to obtain the pCamBarCmpCG and the pCamBarCapG plasmids, respectively. The two constructs are used to transform Agrobacterium tumefaciens cells.

Example 16 Stable Expression in Arabidopsis thaliana

[0212] 1. Plant Production and Transfection

[0213]Arabidopsis thaliana (Columbia 0) wt seeds are sown, cold-treated 3 days at 4° C. in the dark to synchronize germination and transferred to growth room (22° C./24 h light). Germinated plants are infiltrated when they have produced a maximum number of unopened buds. The infiltration is carried out according to the protocol described by Clough and Bent, Plant J. 16: 735-743, 1987.

[0214] 2. Selection of Transgenic Plants

[0215] Seedlings obtained from T1-generation seeds are selected by spraying with the BASTA herbicide (Plüss-Staufer AG/SA) (150 mg/l) every five days for three times. Twenty pCamBarCmpCG and nine pCamBarCapG resistant plants are collected after selection. They are grown under the described condition to obtain the T2-generation seeds. These seeds undergo the same procedure as described above for the wt, the T2 seedlings are selected by BASTA spraying, and the resistant mutants analyzed for GUS-gene expression.

[0216] 3. Histochemical GUS Expression in Stably Transformed Arabidopsis Plants

[0217] Histochemical GUS staining is done as described in Example 11. The analysis is performed in 15 ml culture tubes by immersion of the transformed seedlings in the X-gluc solution, application of 130 mBar pressure for 10 min and incubation at 37° C. overnight. Results obtained from Arabidopsis plants transformed with pCamBarCmpCG indicate a constitutive expression of the GUS reporter gene driven by the CmpC promoter in all vegetative organs.

[0218] 4. Quantitative Enzymatic GUS Assays in Stably Transformed Arabidopsis Plants

[0219] A. Sample Preparation

[0220] Four plants per transgenic line and one leaf per each of these plants are selected for the test. The four leaves are collected in the same eppendorf tube, frozen with liquid nitrogen and ground to fine powder with disposable grinders. This is diluted in 150 μl GUS buffer (0.05M NaPO₄, pH 7, 0.01M EDTA, 0.1% Triton-X-100, 0.1% Sarkosyl), incubated 5 min at 37° C. and spun in a microcentrifuge for 5 min at maximum speed. The supernatant is transferred to a new eppendorf tube and these samples are used for the enzymatic test. Protein in these plant extracts is estimated according to the Bradford method (Bradford, M. M. Anal. Biochem. 72: 248-254, 1976) using BSA as a standard. The presence of the promoter and GUS gene in the mutant lines is confirmed by PCR analysis using the extracted samples as a template.

[0221] B. Fluorometric GUS Assay

[0222] The fluorometric GUS assay is done as described in Example 5. For detection of GUS gene expression samples are diluted in GUS buffer. The reaction is carried out in the presence of an equal amount of GUS-reaction buffer at 37° C. and stopped with 2 M Ammediol. The activity is measured in a Titertek Fluoroskan II.

[0223] Fifteen out of the twenty selected lines transformed with pCamBarCmpCG and eight out of nine lines transformed with pCamBarCapG show GUS enzymatic activity in leaves. The level of activity in several CmpCG transformed lines is comparable to CapG. However the results are variable for the different transgenic lines due to the difference in their genotypes (number of loci and number of transgene insertions per locus).

Example 17 Construction of CmpC Promoter Variants

[0224] All the PCR reactions are carried out with Pfu polymerase (Stratagene) as described in Example 1.

[0225] 1. DNA amplification and cloning Two variants, pCmpMG and pCmpMsG, of the pCmpCG plasmid (Example 3) are produced. The former deletes the sequence “GTGGTCCC” in the CmpC promoter and the latter mutates it to the sequence “GTGCTCGC”.

[0226] To obtain pCmpMG three PCR products are amplified:

[0227] CmpM1 using the Cmp1 forward primer (see Example 3, SEQ ID NO:13), the CmpMr reverse primer (CCATCGTGGTATTTGGTATTG, SEQ ID NO:33) and the pCmpCG plasmid as a template.

[0228] CmpM2 using the CmpMf forward primer (CAATACCAAATACCACGATGG; SEQ ID NO:34), the CmpC2 reverse primer (see Example 3; SEQ ID NO:14) and the pCmpCG plasmid as a template.

[0229] CmpM using the Cmp1 forward primer, the CmpC2 reverse primer and an equimolar mix of CmpM1 and CmpM2 PCR products as a template.

[0230] To obtain pCmpMsG three PCR product are amplified:

[0231] CmpMs1 using the Cmp1 forward primer, the CmpMrs reverse primer (CGTGGTAGCGAGCACTTTGGT, SEQ ID NO:35) and the pCmpCG plasmid as a template.

[0232] CmpMs2 using the CmpMfs forward primer (AAGTGCTCGCTACCACGATGG, SEQ ID NO:36), the Cmp2 reverse primer and the pCmpCG plasmid as a template.

[0233] CmpsM using the Cmp1 forward primer, the Cmp2 reverse primer and an equimolar mix of CmpMs1 and CmpMs2 PCR products as a template.

[0234] To obtain the pCmpMG and the pCmpMsG plasmids the original pCmpCG plasmid is restricted with XbaI and BamHI endonucleases to eliminate the CmpC fragment and to insert in place of it the CmpM and the CmpMs PCR products.

Example 18 Transient Expression Experiments with pCmpM and pCmpMs Constructs

[0235] 1. Suspension Cultures and Protoplast Preparation

[0236] Suspension cultures and protoplasts are produced as described in Example 4.

[0237] 2. GUS Assays

[0238] Transfection and protein extract preparation are carried out as described in the Example 4, and the GUS assay is carried out as described in Example 5.

[0239] GUS assay results are obtained from the average of ten different experiments and normalized to a standard clone value. The deletion of the “GTGGTCCC” sequence in the CmpCG construct reduces expression of the GUS reporter gene to about 50% of the GUS reporter gene expression from CmpC in all protoplast species (see below). The effect induced by the “GTGCTCGC” mutation depends on the plant species. In O. violaceus and O. sativa protoplasts the level of GUS gene expression is decreased to 65.2% and 73.6%, respectively. In N. plumbaginifolia protoplasts the CmpMs promoter activity is similar to the CmpM promoter.

[0240]N. plumbaginifolia CmpCG 100 CmpMG   52 +/− 12% CmpMsG 53.7 +/− 16% CapG 28.3 +/− 14% O. violaceus CmpCG 100 CmpMG 55.2 +/− 12% CmpMsG 73.6 +/− 10% CapG   5 +/− 1%  O. sativa CmpCG 100 CmpMG 59.6 +/− 21% CmpMsG 65.2 +/− 20% CapG 42.5 +/− 16%

Example 19 Construction of Plant Transformation Vectors Containing 5′-promoter Fragments Operably Linked to GFP or GUS Reporter Genes

[0241] 1. Promoter Amplification and Cloning

[0242] A chimeric gene is constructed that includes a DNA sequence from the CmYLCV full-length transcript promoter (SEQ ID NO:1) fused to the plant optimized GFP with intron (synGFPI, SEQ ID NO:37) or GUS reporter gene with intron (GIG; SEQ ID NO:38) sequence. The GIG gene contains the ST-LS1 intron from Solanum tuberosum at nt 385 to nt 576 of SEQ ID NO: 38 (obtained from Dr. Stanton Gelvin, Purdue University, and described in Narasimhulu, et al 1996, Plant Cell, 8: 873-886.). SynGFPI is a plant optimized GFP gene into which the ST-LS1 intron is introduced (nt 278 to nt 465 of SEQ ID NO:37). For the promoters, the CmYLCV genomic DNA is used as a template for the polymerase chain reaction (PCR). Gene specific primers are designed to amplify the DNA sequence. A gene fragment corresponding to CmpC (SEQ ID NO:2) is isolated using the Cmpf-B forward primer (SEQ ID NO:29) in combination with the 5′ to 3′ primer CGCGGATTGCTCCCTTAACAATGAGG (SEQ ID NO:39) as reverse primer. A gene fragment corresponding to CmpS (SEQ ID NO:3) is isolated using the Cmpf-B (SEQ ID NO:29) forward primer in combination with the CmpS-B reverse primer (SEQ ID NO:30). All three primers contain the BamHI restriction enzyme recognition site CGCGGA at their 5′ end. All PCR reactions are carried out with Pfu polymerase according to manufacturers recommendations (Stratagene). A thermocycler (DNA Engine, MJResearch, Inc. Waltham, Mass. USA) is used to amplify the promoter fragments using the following PCR conditions: [(94° C.:10 s):(94° C.:10 s/56° C.:30 s/72° C.:1 min)×20]:(72° C.:1.5 m)]. Following restriction digestion with BamHI the promoter fragments are ligated into pUC-based vectors with the GIG gene or synGFPI gene to create the promoter-reporter gene fusions operatively linked with a nos terminator.

[0243] 2. Produced Promoter-Reporter Cassettes

[0244] CmpS promoter fragment+synGFPI gene+nos (SEQ ID NO:40)

[0245] CmpS promoter fragment+GIG gene+nos (SEQ ID NO:41)

[0246] CmpC promoter fragment+synGFPI gene+nos (SEQ ID NO:42)

[0247] CmpC promoter fragment+GIG gene+nos (SEQ ID NO:43) Gene SEQ ID NO Promoter (synGFPI or GIG) terminator 40 nt 1 to nt 402 nt 411 to nt 1331 nt 1343 to nt 1618 41 nt 1 to nt 405 nt 425 to nt 2425 nt 2479 to nt 2751 42 nt 1 to nt 354 nt 380 to nt 1292 nt 1304 to nt 1577 43 nt 1 to nt 354 nt 399 to nt 2399 nt 2453 to nt 2725

[0248] 3. Subcloning into Agrobacterium Binary Vector

[0249] The promoter-reporter cassettes (SEQ ID NO:40 to 43) containing the promoter fragment, the reporter gene and the nos terminator are inserted into binary vector pNOV2117 for maize and binary vector pNOV4200 for tomato transformation. pNOV2117 (SEQ ID NO:44) is a binary vector with VS1 origin of replication, a copy of the Agrobacterium virG gene in the backbone, and a Maize Ubiquitin promoter-PMI gene-nos terminator expression cassette between the left and right borders of T-DNA. PMI (phosphomannose isomerase) is the coding region of the E. coli manA gene (Joersbo and Okkels, 1996, Plant Cell Reports 16:219-221, Negrotto et al., 2000, Plant Cell Reports 19:798-803). The nos (nopaline synthase) terminator is obtained from Agrobacterium tumefaciens T-DNA (Depicker et al., 1982, J. Mol. Appl. Genet. 1 (6), 561-573). The maize ubiquitin promoter, the phosphomannose isomerase coding region and the nos terminator are located at nt 31 to nt 2012, nt 2109 to nt 3212 and nt 3273 to 3521 respectively, of pNOV2117 (SEQ ID NO:44). The reporter-promoter cassettes are inserted closest to the right border. The selectable marker expression cassette in the binary vectors is closest to the left border.

[0250] For tomato transformation, pNOV4200 (SEQ ID NO:45) is used as the binary vector, which contains the VS1 origin of replication, a copy of the Agrobacterium virG gene in the backbone, and a hygromycin selectable marker cassette between the left and right border sequences. The hygromycin selectable marker cassette comprises the Arabidopsis Ubiquitin 3 promoter (Ubq3(At), Callis et al., J. Biol. Chem. 265:12486-12493 (1990)) operably linked to the gene encoding hygromycin resistance (denoted here as “HYG”, synthetic hygromycin B phosphotransferase gene from E.coli, Patent: JP 1986085192-A 1 30-APR-1986) and the nos terminator (Depicker et al., J. Mol. Appl. Genet. 1 (6), 561-573 (1982). The Arabidopsis ubiquitin promoter, HYG gene and nos terminator are located at nt 162 to nt 11494, nt 1897 to nt 2939 and nt 2939 to nt 3236, respectively of pNOV4200 (SEQ ID NO:45). The reporter-promoter cassettes (SEQ ID NO:40 to 43) are inserted closest to the right border. The selectable marker expression cassette in the binary vectors is closest to the left border.

[0251] Shown below are the orientations of the selectable marker and promoter-reporter cassettes in the binary vector constructs.

[0252] 4. Binary Vector Constructs:

[0253] NOV4215 (RB CmpS promoter fragment+synGFPI gene+nos-ZmUbi+PMI gene+nos LB)

[0254] NOV4216 (RB nos+synGFPI gene+CmpS promoter fragment-ZmUbi+PMI gene+nos LB)

[0255] NOV4217 (RB CmpS promoter fragment+synGFPI gene+nos-Ubq3(At)+HYG gene+nos LB)

[0256] NOV4220 (RB CmpS promoter fragment+GIG gene+nos-Ubq3(At)+HYG gene+nos LB)

[0257] NOV4224 (RB CmpC promoter fragment+GIG gene+nos-ZmUbi+HYG gene+nos LB)

[0258] NOV4226 (RB CmpC promoter fragment+synGFPI gene+nos-ZmUbi+PMI gene+nos LB)

[0259] Additional cassettes are constructed with known promoters to be used for comparison. To this end, a promoter cassette, ZmUbi-GFP-35S term (SEQ ID NO:46), comprising the maize Ubiquitin promoter (Christensen et al. Plant Molec. Biol. 12: 619-632 (1989)) operatively linked with synGFP and the 35S terminator (35S term) is constructed and a promoter cassette comprising the maize ubiquitin promoter operatively linked with the GIG gene and the nos terminator (NOV 3612, SEQ ID NO:47) is also constructed The ZmUbi promoter, GFP gene and 35S terminator are located at nt 1 to nt 2019, nt 2020 to nt 2751 and nt 2876 to nt 2949, respectively of SEQ ID NO:46. The ZmUbi promoter, GIG gene and nos terminator are located at nt 1 to nt 1982, nt 2015 to nt 4015 and nt 4069 to nt 4341, respectively of SEQ ID NO:47. The ZmUbi-GIG nos cassette (SEQ ID NO: 47) is cloned into a pUC-based vector. The ZmUbi-GFP-nos cassette (SEQ ID NO:46) is cloned into the pNOV2117 binary vector for maize transformation, as described above. Promoter cassettes comprising the Arabidopsis Ubiquitin3 promoter (Ubq3(At)) operatively linked to synGFPI and the nos terminator (SEQ ID NO:48) and the Arabidopsis Ubiquitin3 promoter operatively linked to GIG gene and the nos terminator (SEQ ID NO:49) are constructed. The Ubiqutin3 promoter, synGFPI and the nos terminator are located at nt 1 to nt 1332, nt 1738 to nt 2658 and nt 2670 to nt 2943, respectively of SEQ ID NO: 48. The Ubiqutin3 promoter, GIG gene and the nos terminator are located at nt 1 to nt 1335, nt 1746 to nt 3746 and nt 3800 to nt 4072, respectively of SEQ ID NO: 49. As described above, the promoter cassettes are cloned into the pNOV4200 binary vector containing a hygromycin selectable marker cassette for tomato transformation. Shown below are the orientations of the selectable marker and promoter-reporter cassettes in the binary vector constructs.

[0260] 5. Binary Vector Constructs

[0261] NOV2110 (RB ZmUbi Promoter+synGFP gene+35S term-ZmUbi+PMI gene+nos LB)

[0262] NOV4209 (RB Ubq3(At) promoter+synGFPI gene+nos-Ubq3(At)+HYG gene+nos LB)

[0263] NOV4208 (RB Ubq3(At) promoter+GUS-Intron-GUS gene+nos-Ubq3(At)+HYG gene+nos LB)

Example 20 Agrobacterium-mediated Transformation of Maize

[0264] Transformation of immature maize embryos is performed essentially as described in Negrotto et al., (2000) Plant Cell Reports 19: 798-803. For this example, all media constituents are as described in Negrotto et al., supra. However, various media constituents described in the literature may be substituted.

[0265] 1. Transformation Plasmids and Selectable Marker

[0266] The genes used for transformation are cloned into a vector suitable for maize transformation as described in Example 19. Vectors used contain the phosphomannose isomerase (PMI) gene (Negrotto et al. (2000) Plant Cell Reports 19: 798-803).

[0267] 2. Preparation of Agrobacterium tumefaciens

[0268] Agrobacterium strain LBA4404 (pSB1) containing the plant transformation plasmid is grown on YEP (yeast extract (5 g/L), peptone (10 g/L), NaCl (5 g/L), 15 g/l agar, pH 6.8) solid medium for 2 to 4 days at 28° C. Approximately 0.8×10⁹ Agrobacteria are suspended in LS-inf media supplemented with 100 μM acetosyringone (As) (Negrotto et al., (2000) Plant Cell Rep 19: 798-803). Bacteria are pre-induced in this medium for 30-60 minutes.

[0269] 3. Inoculation

[0270] Immature embryos from A188 or other suitable maize genotypes are excised from 8-12 day old ears into liquid LS-inf+100 μM As. Embryos are rinsed once with fresh infection medium. Agrobacterium solution is then added and embryos are vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos are then transferred scutellum side up to LSAs medium and cultured in the dark for two to three days. Subsequently, between 20 and 25 embryos per petri plate are transferred to LSDc medium supplemented with cefotaxime (250 mg/l) and silver nitrate (1.6 mg/l) and cultured in the dark for 28° C. for 10 days.

[0271] 4. Selection of Transformed Cells and Regeneration of Transformed Plants

[0272] Immature embryos producing embryogenic callus are transferred to LSD1M0.5S medium. The cultures are selected on this medium for 6 weeks with a subculture step at 3 weeks. Surviving calli are transferred either to LSD1M0.5S medium to be bulked-up or to Reg1 medium. Following culturing in the light (16 hour light/8 hour dark regiment), green tissues are then transferred to Reg2 medium without growth regulators and incubated for 1-2 weeks. Plantlets are transferred to Magenta GA-7 boxes (Magenta Corp, Chicago Ill.) containing Reg3 medium and grown in the light. Plants that are PCR positive for the promoter-reporter cassette are transferred to soil and grown in the greenhouse.

Example 21 Stable Transformation of Tomato

[0273] Transformation of tomato is performed essentially as described in Meissner et al., The Plant Journal 12:1465-1472, 1997.

[0274] 1. Transformation Plasmids and Selectable Marker

[0275] The genes used for transformation are cloned into a binary vector suitable for tomato transformation as described in Example 19. Vectors contain the hygromycin resistance gene for selection.

[0276] 2. Preparation of Agrobacterium tumefaciens

[0277] Agrobacterium strain GV3101 containing the plant transformation plasmid is grown on LB (yeast extract (5 g/L), peptone (10 g/L), NaCl (5 g/L), 15 g/l agar, pH 6.8) liquid media for 2 days at 22° C. Approximately 0.8×10⁹ Agrobacteria are suspended in KCMS inoculation media (MS salts (4.3 g/l), Myo-Inositol, Thiamine (1.3 ug/ml), 2,4-D (0.2 ug/ml), Kinetin 0.1 ug/ml and 3% Sucrose, pH 5.5) supplemented with 100 μM acetosyringone. Bacteria are pre-induced in this medium for 60 minutes.

[0278] 3. Seeds Sterilization and Germination.

[0279] Seeds of Micro-Tom cultivar of tomato (Meissner et al., The Plant Journal 12: 1465-1472, 1997) and ZTV-840 cultivars are soaked in 20% bleach solution with Tween for 20 min. Seeds are then washed three times with sterile water and plated on TSG media (½ MS saltes, 1% Sucrose, 1% PhytoAgar, pH 5.8) in Magenta boxes (Magenta Corp, Chicago Ill.) for germination. Cotyledons are excised and cotyledon petioles are cut into 5 mm segments 7 to 10 days after germination.

[0280] 4. Inoculation

[0281] Seven to ten day old cotyledons and cotyledonal petioles segments are incubated for 24 hrs on tobacco BY-2 feeder cells plates (MS salts, Myo-inisitol 0.1 mg/ml, Casein Hydrolysate 0.2 mg/ml, Thiamine-HCl 1.3 μg/ml, 3% Sucrose, pH 5.5) prior to inoculation with Agrobacterium.

[0282] Cotyledons are dipped into liquid KCMS Agrobacterium suspension for 5 minutes. Between 20 and 25 cotyledons are then transferred abaxial side upward on the same feeder cell plates and cultured in the dark for two to three days.

[0283] 5. Selection of Transformed Cotyledons and Petiols and Regeneration of Transformed Plants

[0284] Cotyledons and petiole fragments are transferred onto selection media TSM-1 (4.3 g/l MS salts, 1× B5 Vitamins, 2% Sucrose, 1% Glucose, 1 μg/ml Zeatin, 0.05 μg/ml IAA, 5 μg/ml Hygromicine and 250 μg/ml Carbenicillin, pH 5.8) two to three days after inoculation with Agrobacterium in the light. Cotyledons and petiole segments producing embryogenic callus are transferred to TSM-2 medium (MS Salts 4.3 g/1, 1× MS Vitamins, 2% Sucrose, 1 μg/ml Zeatin, 5 μg/ml Hygromicine and 250 μg/ml Carbenicillin, pH 5.8). Surviving calli forming plantlets are transferred to Magenta GA-7 boxes (Magenta Corp, Chicago Ill.) containing TRM medium (4.3 g/l MS salt, 1×MS Vitamins, 2% Sucrose pH 5.8) and grown in the light. After root formation primary transformants are transferred to soil.

Example 22 Green Fluorescent Protein (GFP) Fluorometric Assay

[0285] For detection of synGFP gene expression the fluorometric assay is performed. Leaf discs of stably transformed maize and tomato are frozen on dry ice. Frozen tissue is throughly ground and mixed with 300 μl of GFP Extraction Buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM MgCl2, 10 mM DTT and 0.1% Sarcosyl). The extracts are separated from the leaf debris by centrifugation followed by transfer into 96 well black plate with clear bottom (Costar 3615). Relative fluoresence units (RFU) are measured by Spectra Fluor Plus Plate Reader at 465 nm excitation and 512 nm emission. GFP activity is normalized to total protein measured using BCA Assay (according to Pierce).

Example 23 Expression Analysis of Stably Transformed Maize and Tomato Plants

[0286] 1. Zea mays Transgenic To Plants

[0287] GFP expression is 10 to 20 times higher under the control of the CmpS promoter (NOV4215, NOV4216) compared to the ZmUbi promoter (NOV2110). These results are obtained from leaf samples of 42 independent To lines representing a total of 93 To plants.

[0288] 2. GFP Elisa Assay

[0289] A quantitative sandwich immunoassay is carried out for the detection of green fluorescent protein (GFP). Two commercially available polyclonal antibodies are used. The goat anti-GFP IAP (Rockland, #600-1-1-215) is immunoaffinity purified and the rabbit anti-GFP antibody (Torrey Pines Biolabs, #TP401) is protein A purified. The GFP protein in the plant extract is captured onto the solid phase microtiter by the rabbit antibody. Then a “sandwich” is formed between the solid phase rabbit antibody, the GFP protein, and the secondary goat antibody that is in the well. After a wash step, where unbound secondary antibody is removed, the bound antibody is detected using an alkaline phosphatase-labeled antibody. Substrate for the enzyme is added and color development is measured by reading the absorbance of each well. The reading is then fit to a standard curve to plot the GFP concentrations versus the absorbance synGFP reporter gene GFP Activity ELISA Binary vector (Average) (Average) Cassette RFU/mf protein ng GFP/mg protein NOV4215 (CmpS) 4722.3 657.1 NOV4216 (CmpS) 2795.8 246.1 NOV2110 (ZmUbi) 272.0 41.4

[0290] 2. Lycopersicon esculentum to Plants

[0291] The CmpS fragment of CmYLCV promoter (NOV 4217) results in synGFP expression level that is significantly higher then the expression level of Ubq3 promoter of Arabidopsis (NOV 4209).

[0292] synGFP Reporter Gene

[0293] The results are obtained from evaluating of intensity of synGFP fluorescence by microscopy in callus and primary shoots of 5 independent stably transformed callus lines transformed with the NOV4217 cassette and 2 independent stably transformed callus lines transformed with the NOV4209 cassette. Binary vector Cassette (Promoter) Callus ID GFP Fluorescence Intensity NOV4217 (CmpS) NOV4217-1 +++++ NOV4217 (CmpS) NOV4217-2 ++++ NOV4217 (CmpS) NOV4217-3 ++++ NOV4217 (CmpS) NOV4217-4 +++ NOV4217 (CmpS) NOV4217-5 +++ NOV4209 (Ubq3 (At)) NOV4209-1 + NOV4209 (Ubq3 (At)) NOV4209-2 +

[0294] Gus-Intron-GUS Reporter Gene

[0295] The results are obtained from evaluating of intensity of GUS staining in 11 independent stably transformed callus lines containing the CmpS promoter-reporter cassettes (lines NOV4220-1 through NOV4220-11). Two stably transformed callus lines containing CmpC promoter fragment of CmYLCV promoter (NOV4224-1, NOV4224-2) are evaluated. For comparison two stably transformed callus lines containing the Arabidopsis Ubiquitin 3 (Ubq3) promoter (NOV4208-1, NOV4208-2) are evaluated. Binary vector cassette (Promoter) Callus ID GUS Staining NOV4220 (CmpS) NOV4220-1 ++++ NOV4220 (CmpS) NOV4220-2 +++ NOV4220 (CmpS) NOV4220-3 ++++ NOV4220 (CmpS) NOV4220-4 +++++ NOV4220 (CmpS) NOV4220-5 +++++ NOV4220 (CmpS) NOV4220-6 +++ NOV4220 (CmpS) NOV4220-7 ++++ NOV4220 (CmpS) NOV4220-8 +++++ NOV4220 (CmpS) NOV4220-9 ++++ NOV4220 (CmpS) NOV4220-10 +++ NOV4220 (CmpS) NOV4220-11 ++++ NOV4224 (CmpC) NOV4224-1 ++++ NOV4224 (CmpC) NOV4224-2 +++ NOV4208 (Ubq3 (At)) NOV4208-1 + NOV4208 (Ubq3(At)) NOV4208-2 ++

Example 24 Transient Expression Experiments in Maize

[0296] 1. Embryos Preparation

[0297] Immature embryos from A188 or other suitable maize genotypes are excised from 8-12 day old ears into liquid 2DG4+0.5d (Duncan's D medium modified to contain 20 mg/l glucose and supplemented with 10 g/l mannose) and cultured for 1 to 2 days.

[0298] 2. Plasmolysis

[0299] Immature embryos were incubated in 12% sucrose solution for 3-4 hour prior bombardment. Embryos are arranged in 8-10 mm circles on a plate.

[0300] 3. Bombardment

[0301] Transfection by Particle Bombardment of Maize embryos is performed in the presence of 5 μg of plasmid DNA at 650 psi as described (Wright et al, 2001, Plant Cell Reports, In Press) In case an ‘in press’ reference won't do, here are the details lifted from the paper: Fragment DNA is precipitated onto gold microcarriers (<1 um) as described in the DuPont Biolistics Manual. Genes are delivered to the target tissue cells using the PDS-1000He Biolisitcs device. The settings on the device are as follows: 8 mm between the rupture disk and the macrocarrier, 10 mm between the macrocarrier and the stopping screen and 7 cm between the stopping screen and the target. The plates are shot twice with DNA-coated particles using 650 psi-rupture disks. To reduce tissue damage from the shock wave of the helium blast, a stainless steel mesh, 200 openings per lineal inch horizontally and vertically (McMaster-Carr, New Brunswick, N.J.) is placed between the stopping screen and the target tissue. Embryos are cultivated on the plates for 48 hrs in the dark at 25° C. Protein extracts are prepared by lysing cells in GUS lysis buffer and clarified by centrifugation.

Example 25 GUS Assays

[0302] For detection of GUS gene expression the histochemical and chemiluminescent assays are performed.

[0303] 1. Histochemical β-glucuronidase (GUS) Assay

[0304] Maize embryos and tomato in vitro callus lines are used in GUS assays. Either whole embryos or small pieces of callus are dipped into GUS staining solution. GUS staining solution contains 1 mM 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc, Duchefa, 20 mM stock in DMSO), 100 mM Na-phosphate buffer pH 7.0, 10 mM EDTA pH 8.0, and 0.1% Triton X100. Tissue samples are incubated at 37° C. for 1-16 hours. If necessary, samples are cleared with several washes of 70% EtOH to remove chlorophyll

[0305] 2. β-glucuronidase (GUS) Chemilluminescent Assay

[0306] For quantitive analysis of Gus expression the GUS chemilluminescent assay is performed using GUS-Light Kit (Tropix. Inc) according to manufacturer's instructions.

[0307] The activity is measured in a Luminometer.

[0308] The GUS results with construct pCmpS and pCmpMG (Example 17) are normalized to a comparison clone value (pNOV3612, Example 19). Results obtained from two different experiments show that the 8 bp deletion in CmYLCV promoter (pCmpMG, Example 17) does not significantly affect the expression levels of the GUS reporter as compared to pCmpS.

[0309] Transient Expression in Z. mays Embryos

[0310] GUS Activity at 48 and 72 Hours After Transfection GUS Activity RFU/mg protein Construct ID 48 hr 72 hr pCmpS 103.7 51.7 pCmpMG 81.4 70.8 ZmUbi (pNOV3612) 52.6 70.2

[0311]

1 49 1 670 DNA Cestrum yellow leaf curling virus 1 attactggca gacaaagtgg cagacatact gtcccacaaa tgaagatgga atctgtaaaa 60 gaaaacgcgt gaaataatgc gtctgacaaa ggttaggtcg gctgccttta atcaatacca 120 aagtggtccc taccacgatg gaaaaactgt gcagtcggtt tggctttttc tgacgaacaa 180 ataagattcg tggccgacag gtgggggtcc accatgtgaa ggcatcttca gactccaata 240 atggagcaat gacgtaaggg cttacgaaat aagtaagggt agtttgggaa atgtccactc 300 acccgtcagt ctataaatac ttagcccctc cctcattgtt aagggagcaa aatctcagag 360 agatagtcct agagagagaa agagagcaag tagcctagaa gtagtcaagg cggcgaagta 420 ttcaggcagg gtggccagga agaagaaaag ccaagacgac gaaaacaggt aagagctaag 480 ctttctcatc tcaaagatga ttcttgatga tttttgtctc cacggtccgt ataggatcca 540 ctgaattgat aaatatcata tggtttgtat aaaacccgat atttaaatct gtatcattct 600 gtttgaataa aacttgatac tttgttggag tcgtttgtaa aaacataaac aattataatc 660 tgttaaaaac 670 2 346 DNA Cestrum yellow leaf curling virus 2 ctggcagaca aagtggcaga catactgtcc cacaaatgaa gatggaatct gtaaaagaaa 60 acgcgtgaaa taatgcgtct gacaaaggtt aggtcggctg cctttaatca ataccaaagt 120 ggtccctacc acgatggaaa aactgtgcag tcggtttggc tttttctgac gaacaaataa 180 gattcgtggc cgacaggtgg gggtccacca tgtgaaggca tcttcagact ccaataatgg 240 agcaatgacg taagggctta cgaaataagt aagggtagtt tgggaaatgt ccactcaccc 300 gtcagtctat aaatacttag cccctccctc attgttaagg gagcaa 346 3 400 DNA Cestrum yellow leaf curling virus 3 ctggcagaca aagtggcaga catactgtcc cacaaatgaa gatggaatct gtaaaagaaa 60 acgcgtgaaa taatgcgtct gacaaaggtt aggtcggctg cctttaatca ataccaaagt 120 ggtccctacc acgatggaaa aactgtgcag tcggtttggc tttttctgac gaacaaataa 180 gattcgtggc cgacaggtgg gggtccacca tgtgaaggca tcttcagact ccaataatgg 240 agcaatgacg taagggctta cgaaataagt aagggtagtt tgggaaatgt ccactcaccc 300 gtcagtctat aaatacttag cccctccctc attgttaagg gagcaaaatc tcagagagat 360 agtcctagag agagaaagag agcaagtagc ctagaagtag 400 4 634 DNA Cestrum yellow leaf curling virus 4 ctggcagaca aagtggcaga catactgtcc cacaaatgaa gatggaatct gtaaaagaaa 60 acgcgtgaaa taatgcgtct gacaaaggtt aggtcggctg cctttaatca ataccaaagt 120 ggtccctacc acgatggaaa aactgtgcag tcggtttggc tttttctgac gaacaaataa 180 gattcgtggc cgacaggtgg gggtccacca tgtgaaggca tcttcagact ccaataatgg 240 agcaatgacg taagggctta cgaaataagt aagggtagtt tgggaaatgt ccactcaccc 300 gtcagtctat aaatacttag cccctccctc attgttaagg gagcaaaatc tcagagagat 360 agtcctagag agagaaagag agcaagtagc ctagaagtag tcaaggcggc gaagtattca 420 ggcagggtgg ccaggaagaa gaaaagccaa gacgacgaaa acaggtaaga gctaagcttt 480 ctcatctcaa agatgattct tgatgatttt tgtctccacg gtccgtatag gatccactga 540 attgataaat atcatatggt ttgtataaaa cccgatattt aaatctgtat cattctgttt 600 gaataaaact tgatactttg ttggagtcgt ttgt 634 5 32 DNA Cestrum yellow leaf curling virus 5 aaaaacataa acaattataa tctgttaaaa ac 32 6 104 DNA Cestrum yellow leaf curling virus 6 ggcacagctg tcagttgtgc aaatccgagt catctggacc acaaacatca gaagagggtc 60 tacaagagtc agaagacgaa gacttttcgg tgctagttta atta 104 7 18 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 7 gaccacaaac atcagaag 18 8 18 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 8 caaacttatt gggtaatc 18 9 21 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 9 cagggtacca ctaaaatcac c 21 10 19 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 10 aggggatccc caattcccc 19 11 21 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 11 aggggtacca tcgatatgga g 21 12 19 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 12 ttaggatccg ccctgccac 19 13 21 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 13 cttctagaca aagtggcaga c 21 14 21 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 14 ttggtacctt aacaatgagg g 21 15 21 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 15 ctacttctag gtaccttgct c 21 16 21 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 16 ttggtacctt aacaatgagg g 21 17 20 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 17 ggggatcccc agtctctctc 20 18 18 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 18 gtgaattcga gctcggta 18 19 668 DNA Cestrum yellow leaf curling virus 19 attactggca gacaaagtgg cagacatact gtcccacaaa tgaagatgga atctgtaaaa 60 gaaaacgcgt gaaataatgc gtctgacaaa ggttaggtcg gctgccttta atcaatacca 120 aagtggtccc taccacgatg gaaaaactgt gcagtcggtt tggctttttc tgacgaacaa 180 ataagattcg tggccgacag gtgggggtcc accatgtgaa ggcatcttca gactccaata 240 atggagcaat gacgtaaggg cttacgaaat aagtaagggt agtttgggaa atgtccactc 300 acccgtcagt ctataaatac ttagcccctc cctcattgtt aagggagcaa aatctcagag 360 agatagtcct agagagagaa agagagcaag tagcctagaa gtagtcaagg cggcgaagta 420 ttcaggcagg tggccaggaa gaagaaaagc caagacgacg aaaacaggta agagctaagc 480 tttctcatct caaagatgat tcttgatgat ttttgtctcc acggtccgta taggatcact 540 gaattgataa atatcatatg gtttgtataa aacccgatat ttaaatctgt atcattctgt 600 ttgaataaaa cttgatactt tgttggagtc gtttgtaaaa acataaacaa ttataatctg 660 ttaaaaac 668 20 632 DNA Cestrum yellow leaf curling virus 20 ctggcagaca aagtggcaga catactgtcc cacaaatgaa gatggaatct gtaaaagaaa 60 acgcgtgaaa taatgcgtct gacaaaggtt aggtcggctg cctttaatca ataccaaagt 120 ggtccctacc acgatggaaa aactgtgcag tcggtttggc tttttctgac gaacaaataa 180 gattcgtggc cgacaggtgg gggtccacca tgtgaaggca tcttcagact ccaataatgg 240 agcaatgacg taagggctta cgaaataagt aagggtagtt tgggaaatgt ccactcaccc 300 gtcagtctat aaatacttag cccctccctc attgttaagg gagcaaaatc tcagagagat 360 agtcctagag agagaaagag agcaagtagc ctagaagtag tcaaggcggc gaagtattca 420 ggcaggtggc caggaagaag aaaagccaag acgacgaaaa caggtaagag ctaagctttc 480 tcatctcaaa gatgattctt gatgattttt gtctccacgg tccgtatagg atcactgaat 540 tgataaatat catatggttt gtataaaacc cgatatttaa atctgtatca ttctgtttga 600 ataaaacttg atactttgtt ggagtcgttt gt 632 21 28 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 21 cccgctgcag atcgttcaaa catttggc 28 22 27 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 22 cccgaagctt tctagagatc tagtaac 27 23 41 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 23 gggcggatcc gcatgcatgt cttaaggtaa gctcactaag g 41 24 34 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 24 ccgcgctgca ggctgctttc aagcaagttc tgcg 34 25 35 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 25 ttgagagctc gtttaattac tggcagacaa agtgg 35 26 34 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 26 ttgactgcag gtttatgttt ttacaaacga ctcc 34 27 137 DNA Artificial Sequence Description of Artificial Sequence AGLINK multicloning site 27 gcggccgctc cggattcgaa ttaattaacg tacgaagctt gcatgcctgc agtgatcacc 60 atggtcgact ctagaggatc cccgggtacc gagctcgaat tcggcgcgcc caattgattt 120 aaatggccgc tgcggcc 137 28 216 DNA Artificial Sequence Description of Artificial Sequence BIGLINK multicloning site 28 ggccgcagcg gccatttaaa tcaattgggc gcgccgaatt cgagctcggt acccggggat 60 cctctagagt cgaccatggt gatcactgca ggcatgcaag cttcgtacgt taattaattc 120 gaatccggag cggccgcacg cgtgggcccg tttaaacctc gagagatctg ctagccctgc 180 aggaaattta ccggtgcccg ggcggccagc atggcc 216 29 28 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 29 cgcggatcct ggcagacaaa gtggcaga 28 30 26 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 30 cgcggatcct acttctaggc tacttg 26 31 7195 DNA Artificial Sequence Description of Artificial Sequence vector pNOV2804 31 gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60 aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120 acaagccgtt ttacgtttgg aactgacaga accgcaacgc tgcaggaatt ggccgcagcg 180 gccatttaaa tcaattgggc gcgtacgtag cactagtgcg cgatcgctta attaagcggc 240 gcgcctaaag cttgcatgcc tgcaggtcga ctctagagga tccccgatca tgcaaaaact 300 cattaactca gtgcaaaact atgcctgggg cagcaaaacg gcgttgactg aactttatgg 360 tatggaaaat ccgtccagcc agccgatggc cgagctgtgg atgggcgcac atccgaaaag 420 cagttcacga gtgcagaatg ccgccggaga tatcgtttca ctgcgtgatg tgattgagag 480 tgataaatcg actctgctcg gagaggccgt tgccaaacgc tttggcgaac tgcctttcct 540 gttcaaagta ttatgcgcag cacagccact ctccattcag gttcatccaa acaaacacaa 600 ttctgaaatc ggttttgcca aagaaaatgc cgcaggtatc ccgatggatg ccgccgagcg 660 taactataaa gatcctaacc acaagccgga gctggttttt gcgctgacgc ctttccttgc 720 gatgaacgcg tttcgtgaat tttccgagat tgtctcccta ctccagccgg tcgcaggtgc 780 acatccggcg attgctcact ttttacaaca gcctgatgcc gaacgtttaa gcgaactgtt 840 cgccagcctg ttgaatatgc agggtgaaga aaaatcccgc gcgctggcga ttttaaaatc 900 ggccctcgat agccagcagg gtgaaccgtg gcaaacgatt cgtttaattt ctgaatttta 960 cccggaagac agcggtctgt tctccccgct attgctgaat gtggtgaaat tgaaccctgg 1020 cgaagcgatg ttcctgttcg ctgaaacacc gcacgcttac ctgcaaggcg tggcgctgga 1080 agtgatggca aactccgata acgtgctgcg tgcgggtctg acgcctaaat acattgatat 1140 tccggaactg gttgccaatg tgaaattcga agccaaaccg gctaaccagt tgttgaccca 1200 gccggtgaaa caaggtgcag aactggactt cccgattcca gtggatgatt ttgccttctc 1260 gctgcatgac cttagtgata aagaaaccac cattagccag cagagtgccg ccattttgtt 1320 ctgcgtcgaa ggcgatgcaa cgttgtggaa aggttctcag cagttacagc ttaaaccggg 1380 tgaatcagcg tttattgccg ccaacgaatc accggtgact gtcaaaggcc acggccgttt 1440 agcgcgtgtt tacaacaagc tgtaagagct tactgaaaaa attaacatct cttgctaagc 1500 tgggagctct agatccccga atttccccga tcgttcaaac atttggcaat aaagtttctt 1560 aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt 1620 taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat 1680 tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta 1740 ggataaatta tcgcgcgcgg tgtcatctat gttactagat cgggaattgg gtaccatgcc 1800 cgggcggcca gcatggccgt atccgcaatg tgttattaag ttgtctaagc gtcaatttgt 1860 ttacaccaca atatatcctg ccaccagcca gccaacagct ccccgaccgg cagctcggca 1920 caaaatcacc actcgataca ggcagcccat cagaattaat tctcatgttt gacagcttat 1980 catcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat 2040 ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg 2100 gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa atgagctgtt 2160 gacaattaat catccggctc gtataatgtg tggaattgtg agcggataac aatttcacac 2220 aggaaacaga ccatgaggga agcgttgatc gccgaagtat cgactcaact atcagaggta 2280 gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt gtacggctcc 2340 gcagtggatg gcggcctgaa gccacacagt gatattgatt tgctggttac ggtgaccgta 2400 aggcttgatg aaacaacgcg gcgagctttg atcaacgacc ttttggaaac ttcggcttcc 2460 cctggagaga gcgagattct ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc 2520 attccgtggc gttatccagc taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac 2580 attcttgcag gtatcttcga gccagccacg atcgacattg atctggctat cttgctgaca 2640 aaagcaagag aacatagcgt tgccttggta ggtccagcgg cggaggaact ctttgatccg 2700 gttcctgaac aggatctatt tgaggcgcta aatgaaacct taacgctatg gaactcgccg 2760 cccgactggg ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc 2820 gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat ggagcgcctg 2880 ccggcccagt atcagcccgt catacttgaa gctaggcagg cttatcttgg acaagaagat 2940 cgcttggcct cgcgcgcaga tcagttggaa gaatttgttc actacgtgaa aggcgagatc 3000 accaaagtag tcggcaaata aagctctagt ggatctccgt acccccgggg gatctggctc 3060 gcggcggacg cacgacgccg gggcgagacc ataggcgatc tcctaaatca atagtagctg 3120 taacctcgaa gcgtttcact tgtaacaacg attgagaatt tttgtcataa aattgaaata 3180 cttggttcgc atttttgtca tccgcggtca gccgcaattc tgacgaactg cccatttagc 3240 tggagatgat tgtacatcct tcacgtgaaa atttctcaag cgctgtgaac aagggttcag 3300 attttagatt gaaaggtgag ccgttgaaac acgttcttct tgtcgatgac gacgtcgcta 3360 tgcggcatct tattattgaa taccttacga tccacgcctt caaagtgacc gcggtagccg 3420 acagcaccca gttcacaaga gtactctctt ccgcgacggt cgatgtcgtg gttgttgatc 3480 taaatttagg tcgtgaagat gggctcgaga tcgttcgtaa tctggcggca aagtctgata 3540 ttccaatcat aattatcagt ggcgaccgcc ttgaggagac ggataaagtt gttgcactcg 3600 agctaggagc aagtgatttt atcgctaagc cgttcagtat cagagagttt ctagcacgca 3660 ttcgggttgc cttgcgcgtg cgccccaacg ttgtccgctc caaagaccga cggtcttttt 3720 gttttactga ctggacactt aatctcaggc aacgtcgctt gatgtccgaa gctggcggtg 3780 aggtgaaact tacggcaggt gagttcaatc ttctcctcgc gtttttagag aaaccccgcg 3840 acgttctatc gcgcgagcaa cttctcattg ccagtcgagt acgcgacgag gaggtttatg 3900 acaggagtat agatgttctc attttgaggc tgcgccgcaa acttgaggca gatccgtcaa 3960 gccctcaact gataaaaaca gcaagaggtg ccggttattt ctttgacgcg gacgtgcagg 4020 tttcgcacgg ggggacgatg gcagcctgag ccaattccca gatccccgag gaatcggcgt 4080 gagcggtcgc aaaccatccg gcccggtaca aatcggcgcg gcgctgggtg atgacctggt 4140 ggagaagttg aaggccgcgc aggccgccca gcggcaacgc atcgaggcag aagcacgccc 4200 cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa gaatcccggc aaccgccggc 4260 agccggtgcg ccgtcgatta ggaagccgcc caagggcgac gagcaaccag attttttcgt 4320 tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc atcatggacg tggccgtttt 4380 ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc cgctacgagc ttccagacgg 4440 gcacgtagag gtttccgcag ggccggccgg catggccagt gtgtgggatt acgacctggt 4500 actgatggcg gtttcccatc taaccgaatc catgaaccga taccgggaag ggaagggaga 4560 caagcccggc cgcgtgttcc gtccacacgt tgcggacgta ctcaagttct gccggcgagc 4620 cgatggcgga aagcagaaag acgacctggt agaaacctgc attcggttaa acaccacgca 4680 cgttgccatg cagcgtacga agaaggccaa gaacggccgc ctggtgacgg tatccgaggg 4740 tgaagccttg attagccgct acaagatcgt aaagagcgaa accgggcggc cggagtacat 4800 cgagatcgag ctagctgatt ggatgtaccg cgagatcaca gaaggcaaga acccggacgt 4860 gctgacggtt caccccgatt actttttgat cgatcccggc atcggccgtt ttctctaccg 4920 cctggcacgc cgcgccgcag gcaaggcaga agccagatgg ttgttcaaga cgatctacga 4980 acgcagtggc agcgccggag agttcaagaa gttctgtttc accgtgcgca agctgatcgg 5040 gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg gggcaggctg gcccgatcct 5100 agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc gccggttcct aatgtacgga 5160 gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt cgaaaaggtc tctttcctgt 5220 ggatagcacg tacattggga acccaaagcc gtacattggg aaccggaacc cgtacattgg 5280 gaacccaaag ccgtacattg ggaaccggtc acacatgtaa gtgactgata taaaagagaa 5340 aaaaggcgat ttttccgcct aaaactcttt aaaacttatt aaaactctta aaacccgcct 5400 ggcctgtgca taactgtctg gccagcgcac agccgaagag ctgcaaaaag cgcctaccct 5460 tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg cctatcgcgg ccgctggccg 5520 ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg cgcggacaag ccgcgccgtc 5580 gccactcgac cgccggcgct gaggtctgcc tcgtgaagaa ggtgttgctg actcatacca 5640 ggcctgaatc gccccatcat ccagccagaa agtgagggag ccacggttga tgagagcttt 5700 gttgtaggtg gaccagttgg tgattttgaa cttttgcttt gccacggaac ggtctgcgtt 5760 gtcgggaaga tgcgtgatct gatccttcaa ctcagcaaaa gttcgattta ttcaacaaag 5820 ccgccgtccc gtcaagtcag cgtaatgctc tgccagtgtt acaaccaatt aaccaattct 5880 gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca 5940 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc 6000 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa 6060 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg 6120 actgaatccg gtgagaatgg caaaagctct gcattaatga atcggccaac gcgcggggag 6180 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 6240 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 6300 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 6360 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 6420 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 6480 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 6540 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 6600 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 6660 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 6720 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 6780 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 6840 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 6900 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 6960 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 7020 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 7080 tttgatccgg aattaattcc tgtggttggc atgcacatac aaatggacga acggataaac 7140 cttttcacgc ccttttaaat atccgattat tctaataaac gctcttttct cttag 7195 32 4224 DNA Artificial Sequence Description of Artificial Sequence vector pNOV3604 32 agcttgcatg cctgcaggtc gactctagag gatccccgat catgcaaaaa ctcattaact 60 cagtgcaaaa ctatgcctgg ggcagcaaaa cggcgttgac tgaactttat ggtatggaaa 120 atccgtccag ccagccgatg gccgagctgt ggatgggcgc acatccgaaa agcagttcac 180 gagtgcagaa tgccgccgga gatatcgttt cactgcgtga tgtgattgag agtgataaat 240 cgactctgct cggagaggcc gttgccaaac gctttggcga actgcctttc ctgttcaaag 300 tattatgcgc agcacagcca ctctccattc aggttcatcc aaacaaacac aattctgaaa 360 tcggttttgc caaagaaaat gccgcaggta tcccgatgga tgccgccgag cgtaactata 420 aagatcctaa ccacaagccg gagctggttt ttgcgctgac gcctttcctt gcgatgaacg 480 cgtttcgtga attttccgag attgtctccc tactccagcc ggtcgcaggt gcacatccgg 540 cgattgctca ctttttacaa cagcctgatg ccgaacgttt aagcgaactg ttcgccagcc 600 tgttgaatat gcagggtgaa gaaaaatccc gcgcgctggc gattttaaaa tcggccctcg 660 atagccagca gggtgaaccg tggcaaacga ttcgtttaat ttctgaattt tacccggaag 720 acagcggtct gttctccccg ctattgctga atgtggtgaa attgaaccct ggcgaagcga 780 tgttcctgtt cgctgaaaca ccgcacgctt acctgcaagg cgtggcgctg gaagtgatgg 840 caaactccga taacgtgctg cgtgcgggtc tgacgcctaa atacattgat attccggaac 900 tggttgccaa tgtgaaattc gaagccaaac cggctaacca gttgttgacc cagccggtga 960 aacaaggtgc agaactggac ttcccgattc cagtggatga ttttgccttc tcgctgcatg 1020 accttagtga taaagaaacc accattagcc agcagagtgc cgccattttg ttctgcgtcg 1080 aaggcgatgc aacgttgtgg aaaggttctc agcagttaca gcttaaaccg ggtgaatcag 1140 cgtttattgc cgccaacgaa tcaccggtga ctgtcaaagg ccacggccgt ttagcgcgtg 1200 tttacaacaa gctgtaagag cttactgaaa aaattaacat ctcttgctaa gctgggagct 1260 ctagatcccc gaatttcccc gatcgttcaa acatttggca ataaagtttc ttaagattga 1320 atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg 1380 taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc 1440 cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat 1500 tatcgcgcgc ggtgtcatct atgttactag atcgggaatt gggtaccgaa ttcactggcc 1560 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 1620 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 1680 caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat 1740 ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca 1800 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 1860 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 1920 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 1980 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 2040 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 2100 agacaataac cctgataaat gcttcaatgg cgcgccgcgg ccgcttaaga atattgaaaa 2160 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 2220 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag 2280 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt 2340 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 2400 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 2460 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 2520 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg 2580 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta 2640 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 2700 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt 2760 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 2820 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 2880 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 2940 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag 3000 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt 3060 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 3120 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 3180 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 3240 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 3300 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 3360 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 3420 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 3480 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 3540 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 3600 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 3660 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 3720 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 3780 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 3840 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 3900 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 3960 gaagcggaag agcttaagcg gccgcggcgc gccgcccaat acgcaaaccg cctctccccg 4020 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca 4080 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact 4140 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 4200 acagctatga ccatgattac gcca 4224 33 21 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 33 ccatcgtggt atttggtatt g 21 34 21 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 34 caataccaaa taccacgatg g 21 35 21 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 35 cgtggtagcg agcactttgg t 21 36 21 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 36 aagtgctcgc taccacgatg g 21 37 912 DNA Artificial artificial sequence synthetic GFP gene with intron 37 atgagcaagg gcgaggagct gttcaccggc gtggtgccaa tcctggtgga gctggacggc 60 gacgtgaacg gccacaagtt cagcgtgagc ggcgagggcg agggcgacgc gacctacggc 120 aagctgaccc tgaagttcat ctgtaccacc ggcaagctcc cggtcccgtg gccgaccctg 180 gtgaccacct tcacctacgg cgtgcagtgt ttcagccgct acccggacca catgaagcgc 240 cacgacttct tcaagagcgc catgccggag ggctacgtaa gtttctgctt ctacctttga 300 tatatatata ataattatca ttaattagta gtaatataat atttcaaata tttttttcaa 360 aataaaagaa tgtagtatat agcaattgct tttctgtagt ttataagtgt gtatatttta 420 atttataact tttctaatat atgaccaaaa tttgttgatg tgcaggtgca ggagcgcacc 480 atcagcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 540 acactagtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 600 ggccacaagc tggagtacaa ctacaacagc cacaacgtgt acatcaccgc ggacaagcag 660 aagaacggca tcaaggcgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag 720 ctggccgacc actaccagca gaacaccccg atcggcgacg gtcctgtgct gctgccggac 780 aaccactacc tgagcaccca gagcgccctg agcaaggacc cgaacgagaa gcgcgaccac 840 atggtgctgc tggagttcgt gaccgccgcc ggcatcaccc acggcatgga cgagctgtac 900 aaggttaact ag 912 38 2001 DNA Artificial artificial sequence GUS gene with intron 38 atggtccgtc ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg cctgtgggca 60 ttcagtctgg atcgcgaaaa ctgtggaatt gatcagcgtt ggtgggaaag cgcgttacaa 120 gaaagccggg caattgctgt gccaggcagt tttaacgatc agttcgccga tgcagatatt 180 cgtaattatg cgggcaacgt ctggtatcag cgcgaagtct ttataccgaa aggttgggca 240 ggccagcgta tcgtgctgcg tttcgatgcg gtcactcatt acggcaaagt gtgggtcaat 300 aatcaggaag tgatggagca tcagggcggc tatacgccat ttgaagccga tgtcacgccg 360 tatgttattg ccgggaaaag tgtacgtaag tttctgcttc tacctttgat atatatataa 420 taattatcat taattagtag taatataata tttcaaatat ttttttcaaa ataaaagaat 480 gtagtatata gcaattgctt ttctgtagtt tataagtgtg tatattttaa tttataactt 540 ttctaatata tgaccaaaat ttgttgatgt gcaggtatca ccgtttgtgt gaacaacgaa 600 ctgaactggc agactatccc gccgggaatg gtgattaccg acgaaaacgg caagaaaaag 660 cagtcttact tccatgattt ctttaactat gccggaatcc atcgcagcgt aatgctctac 720 accacgccga acacctgggt ggacgatatc accgtggtga cgcatgtcgc gcaagactgt 780 aaccacgcgt ctgttgactg gcaggtggtg gccaatggtg atgtcagcgt tgaactgcgt 840 gatgcggatc aacaggtggt tgcaactgga caaggcacta gcgggacttt gcaagtggtg 900 aatccgcacc tctggcaacc gggtgaaggt tatctctatg aactgtgcgt cacagccaaa 960 agccagacag agtgtgatat ctacccgctt cgcgtcggca tccggtcagt ggcagtgaag 1020 ggcgaacagt tcctgattaa ccacaaaccg ttctacttta ctggctttgg tcgtcatgaa 1080 gatgcggact tgcgtggcaa aggattcgat aacgtgctga tggtgcacga ccacgcatta 1140 atggactgga ttggggccaa ctcctaccgt acctcgcatt acccttacgc tgaagagatg 1200 ctcgactggg cagatgaaca tggcatcgtg gtgattgatg aaactgctgc tgtcggcttt 1260 aacctctctt taggcattgg tttcgaagcg ggcaacaagc cgaaagaact gtacagcgaa 1320 gaggcagtca acggggaaac tcagcaagcg cacttacagg cgattaaaga gctgatagcg 1380 cgtgacaaaa accacccaag cgtggtgatg tggagtattg ccaacgaacc ggatacccgt 1440 ccgcaaggtg cacgggaata tttcgcgcca ctggcggaag caacgcgtaa actcgacccg 1500 acgcgtccga tcacctgcgt caatgtaatg ttctgcgacg ctcacaccga taccatcagc 1560 gatctctttg atgtgctgtg cctgaaccgt tattacggat ggtatgtcca aagcggcgat 1620 ttggaaacgg cagagaaggt actggaaaaa gaacttctgg cctggcagga gaaactgcat 1680 cagccgatta tcatcaccga atacggcgtg gatacgttag ccgggctgca ctcaatgtac 1740 accgacatgt ggagtgaaga gtatcagtgt gcatggctgg atatgtatca ccgcgtcttt 1800 gatcgcgtca gcgccgtcgt cggtgaacag gtatggaatt tcgccgattt tgcgacctcg 1860 caaggcatat tgcgcgttgg cggtaacaag aaagggatct tcactcgcga ccgcaaaccg 1920 aagtcggcgg cttttctgct gcaaaaacgc tggactggca tgaacttcgg tgaaaaaccg 1980 cagcagggag gcaaacaatg a 2001 39 26 DNA Artificial artificial sequence PCR primer 39 cgcggattgc tcccttaaca atgagg 26 40 1618 DNA Artificial artificial sequence CmpS-synGFPI-nos expression cassette 40 tcctggcaga caaagtggca gacatactgt cccacaaatg aagatggaat ctgtaaaaga 60 aaacgcgtga aataatgcgt ctgacaaagg ttaggtcggc tgcctttaat caataccaaa 120 gtggtcccta ccacgatgga aaaactgtgc agtcggtttg gctttttctg acgaacaaat 180 aagattcgtg gccgacaggt gggggtccac catgtgaagg catcttcaga ctccaataat 240 ggagcaatga cgtaagggct tacgaaataa gtaagggtag tttgggaaat gtccactcac 300 ccgtcagtct ataaatactt agcccctccc tcattgttaa gggagcaaaa tctcagagag 360 atagtcctag agagagaaag agagcaagta gcctagaagt aggatccacc atgctgcaga 420 tgagcaaggg cgaggagctg ttcaccggcg tggtgccaat cctggtggag ctggacggcg 480 acgtgaacgg ccacaagttc agcgtgagcg gcgagggcga gggcgacgcg acctacggca 540 agctgaccct gaagttcatc tgtaccaccg gcaagctccc ggtcccgtgg ccgaccctgg 600 tgaccacctt cacctacggc gtgcagtgtt tcagccgcta cccggaccac atgaagcgcc 660 acgacttctt caagagcgcc atgccggagg gctacgtaag tttctgcttc tacctttgat 720 atatatataa taattatcat taattagtag taatataata tttcaaatat ttttttcaaa 780 ataaaagaat gtagtatata gcaattgctt ttctgtagtt tataagtgtg tatattttaa 840 tttataactt ttctaatata tgaccaaaat ttgttgatgt gcaggtgcag gagcgcacca 900 tcagcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca 960 cactagtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg 1020 gccacaagct ggagtacaac tacaacagcc acaacgtgta catcaccgcg gacaagcaga 1080 agaacggcat caaggcgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc 1140 tggccgacca ctaccagcag aacaccccga tcggcgacgg tcctgtgctg ctgccggaca 1200 accactacct gagcacccag agcgccctga gcaaggaccc gaacgagaag cgcgaccaca 1260 tggtgctgct ggagttcgtg accgccgccg gcatcaccca cggcatggac gagctgtaca 1320 aggttaacta gagctcaaga tcccccgaat ttccccgatc gttcaaacat ttggcaataa 1380 agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatcta atttctgttg 1440 aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt 1500 tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc 1560 gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcc gggaattg 1618 41 2730 DNA Artificial artificial sequence CmpS-GIG-nos expression cassette 41 aggatcctgg cagacaaagt ggcagacata ctgtcccaca aatgaagatg gaatctgtaa 60 aagaaaacgc gtgaaataat gcgtctgaca aaggttaggt cggctgcctt taatcaatac 120 caaagtggtc cctaccacga tggaaaaact gtgcagtcgg tttggctttt tctgacgaac 180 aaataagatt cgtggccgac aggtgggggt ccaccatgtg aaggcatctt cagactccaa 240 taatggagca atgacgtaag ggcttacgaa ataagtaagg gtagtttggg aaatgtccac 300 tcacccgtca gtctataaat acttagcccc tccctcattg ttaagggagc aaaatctcag 360 agagatagtc ctagagagag aaagagagca agtagcctag aagtaggatc ccctcgaggt 420 cgaccatggt ccgtcctgta gaaaccccaa cccgtgaaat caaaaaactc gacggcctgt 480 gggcattcag tctggatcgc gaaaactgtg gaattgatca gcgttggtgg gaaagcgcgt 540 tacaagaaag ccgggcaatt gctgtgccag gcagttttaa cgatcagttc gccgatgcag 600 atattcgtaa ttatgcgggc aacgtctggt atcagcgcga agtctttata ccgaaaggtt 660 gggcaggcca gcgtatcgtg ctgcgtttcg atgcggtcac tcattacggc aaagtgtggg 720 tcaataatca ggaagtgatg gagcatcagg gcggctatac gccatttgaa gccgatgtca 780 cgccgtatgt tattgccggg aaaagtgtac gtaagtttct gcttctacct ttgatatata 840 tataataatt atcattaatt agtagtaata taatatttca aatatttttt tcaaaataaa 900 agaatgtagt atatagcaat tgcttttctg tagtttataa gtgtgtatat tttaatttat 960 aacttttcta atatatgacc aaaatttgtt gatgtgcagg tatcaccgtt tgtgtgaaca 1020 acgaactgaa ctggcagact atcccgccgg gaatggtgat taccgacgaa aacggcaaga 1080 aaaagcagtc ttacttccat gatttcttta actatgccgg aatccatcgc agcgtaatgc 1140 tctacaccac gccgaacacc tgggtggacg atatcaccgt ggtgacgcat gtcgcgcaag 1200 actgtaacca cgcgtctgtt gactggcagg tggtggccaa tggtgatgtc agcgttgaac 1260 tgcgtgatgc ggatcaacag gtggttgcaa ctggacaagg cactagcggg actttgcaag 1320 tggtgaatcc gcacctctgg caaccgggtg aaggttatct ctatgaactg tgcgtcacag 1380 ccaaaagcca gacagagtgt gatatctacc cgcttcgcgt cggcatccgg tcagtggcag 1440 tgaagggcga acagttcctg attaaccaca aaccgttcta ctttactggc tttggtcgtc 1500 atgaagatgc ggacttgcgt ggcaaaggat tcgataacgt gctgatggtg cacgaccacg 1560 cattaatgga ctggattggg gccaactcct accgtacctc gcattaccct tacgctgaag 1620 agatgctcga ctgggcagat gaacatggca tcgtggtgat tgatgaaact gctgctgtcg 1680 gctttaacct ctctttaggc attggtttcg aagcgggcaa caagccgaaa gaactgtaca 1740 gcgaagaggc agtcaacggg gaaactcagc aagcgcactt acaggcgatt aaagagctga 1800 tagcgcgtga caaaaaccac ccaagcgtgg tgatgtggag tattgccaac gaaccggata 1860 cccgtccgca aggtgcacgg gaatatttcg cgccactggc ggaagcaacg cgtaaactcg 1920 acccgacgcg tccgatcacc tgcgtcaatg taatgttctg cgacgctcac accgatacca 1980 tcagcgatct ctttgatgtg ctgtgcctga accgttatta cggatggtat gtccaaagcg 2040 gcgatttgga aacggcagag aaggtactgg aaaaagaact tctggcctgg caggagaaac 2100 tgcatcagcc gattatcatc accgaatacg gcgtggatac gttagccggg ctgcactcaa 2160 tgtacaccga catgtggagt gaagagtatc agtgtgcatg gctggatatg tatcaccgcg 2220 tctttgatcg cgtcagcgcc gtcgtcggtg aacaggtatg gaatttcgcc gattttgcga 2280 cctcgcaagg catattgcgc gttggcggta acaagaaagg gatcttcact cgcgaccgca 2340 aaccgaagtc ggcggctttt ctgctgcaaa aacgctggac tggcatgaac ttcggtgaaa 2400 aaccgcagca gggaggcaaa caatgaatca acaactctcc tggcgcacca tcgtcggcta 2460 cagcctcggg aattagatcc ccgaatttcc ccgatcgttc aaacatttgg caataaagtt 2520 tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt ctgttgaatt 2580 acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga tgggttttta 2640 tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata tagcgcgcaa 2700 actaggataa attatcgcgc gcggtgtcat 2730 42 1577 DNA Artificial artificial sequence CmpC-synGFPI-nos expression cassette 42 tggcagacaa agtggcagac atactgtccc acaaatgaag atggaatctg taaaagaaaa 60 cgcgtgaaat aatgcgtctg acaaaggtta ggtcggctgc ctttaatcaa taccaaagtg 120 gtccctacca cgatggaaaa actgtgcagt cggtttggct ttttctgacg aacaaataag 180 attcgtggcc gacaggtggg ggtccaccat gtgaaggcat cttcagactc caataatgga 240 gcaatgacgt aagggcttac gaaataagta agggtagttt gggaaatgtc cactcacccg 300 tcagtctata aatacttagc ccctccctca ttgttaaggg agcaacgatc cgcgaagggc 360 gaattcgttt aaacctgcag atgagcaagg gcgaggagct gttcaccggc gtggtgccaa 420 tcctggtgga gctggacggc gacgtgaacg gccacaagtt cagcgtgagc ggcgagggcg 480 agggcgacgc gacctacggc aagctgaccc tgaagttcat ctgtaccacc ggcaagctcc 540 cggtcccgtg gccgaccctg gtgaccacct tcacctacgg cgtgcagtgt ttcagccgct 600 acccggacca catgaagcgc cacgacttct tcaagagcgc catgccggag ggctacgtaa 660 gtttctgctt ctacctttga tatatatata ataattatca ttaattagta gtaatataat 720 atttcaaata tttttttcaa aataaaagaa tgtagtatat agcaattgct tttctgtagt 780 ttataagtgt gtatatttta atttataact tttctaatat atgaccaaaa tttgttgatg 840 tgcaggtgca ggagcgcacc atcagcttca aggacgacgg caactacaag acccgcgccg 900 aggtgaagtt cgagggcgac acactagtga accgcatcga gctgaagggc atcgacttca 960 aggaggacgg caacatcctg ggccacaagc tggagtacaa ctacaacagc cacaatgtgt 1020 acatcaccgc ggacaagcag aagaacggca tcaaggcgaa cttcaagatc cgccacaata 1080 tcgaggacgg cagcgtgcag ctggccgacc actaccagca gaacaccccg atcggcgacg 1140 gtcctgtgct gctgccggac aaccactacc tgagcaccca gagcgccctg agcaaggacc 1200 cgaacgagaa gcgcgaccac atggtgctgc tggagttcgt gaccgccgcc ggcatcaccc 1260 acggcatgga cgagctgtac aaggttaact agagctctag atccccgaat ttccccgatc 1320 gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga 1380 ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga 1440 cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga 1500 tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt 1560 tactagatcg ggaattg 1577 43 2725 DNA Artificial artificial sequence CmpC-GIG-nos expression cassette 43 tggcagacaa agtggcagac atactgtccc acaaatgaag atggaatctg taaaagaaaa 60 cgcgtgaaat aatgcgtctg acaaaggtta ggtcggctgc ctttaatcaa taccaaagtg 120 gtccctacca cgatggaaaa actgtgcagt cggtttggct ttttctgacg aacaaataag 180 attcgtggcc gacaggtggg ggtccaccat gtgaaggcat cttcagactc caataatgga 240 gcaatgacgt aagggcttac gaaataagta agggtagttt gggaaatgtc cactcacccg 300 tcagtctata aatacttagc ccctccctca ttgttaaggg agcaacgatc cgcgaagggc 360 gaattcctgc agcccggggg atcccctcga ggtcgaccat ggtccgtcct gtagaaaccc 420 caacccgtga aatcaaaaaa ctcgacggcc tgtgggcatt cagtctggat cgcgaaaact 480 gtggaattga tcagcgttgg tgggaaagcg cgttacaaga aagccgggca attgctgtgc 540 caggcagttt taacgatcag ttcgccgatg cagatattcg taattatgcg ggcaacgtct 600 ggtatcagcg cgaagtcttt ataccgaaag gttgggcagg ccagcgtatc gtgctgcgtt 660 tcgatgcggt cactcattac ggcaaagtgt gggtcaataa tcaggaagtg atggagcatc 720 agggcggcta tacgccattt gaagccgatg tcacgccgta tgttattgcc gggaaaagtg 780 tacgtaagtt tctgcttcta cctttgatat atatataata attatcatta attagtagta 840 atataatatt tcaaatattt ttttcaaaat aaaagaatgt agtatatagc aattgctttt 900 ctgtagttta taagtgtgta tattttaatt tataactttt ctaatatatg accaaaattt 960 gttgatgtgc aggtatcacc gtttgtgtga acaacgaact gaactggcag actatcccgc 1020 cgggaatggt gattaccgac gaaaacggca agaaaaagca gtcttacttc catgatttct 1080 ttaactatgc cggaatccat cgcagcgtaa tgctctacac cacgccgaac acctgggtgg 1140 acgatatcac cgtggtgacg catgtcgcgc aagactgtaa ccacgcgtct gttgactggc 1200 aggtggtggc caatggtgat gtcagcgttg aactgcgtga tgcggatcaa caggtggttg 1260 caactggaca aggcactagc gggactttgc aagtggtgaa tccgcacctc tggcaaccgg 1320 gtgaaggtta tctctatgaa ctgtgcgtca cagccaaaag ccagacagag tgtgatatct 1380 acccgcttcg cgtcggcatc cggtcagtgg cagtgaaggg cgaacagttc ctgattaacc 1440 acaaaccgtt ctactttact ggctttggtc gtcatgaaga tgcggacttg cgtggcaaag 1500 gattcgataa cgtgctgatg gtgcacgacc acgcattaat ggactggatt ggggccaact 1560 cctaccgtac ctcgcattac ccttacgctg aagagatgct cgactgggca gatgaacatg 1620 gcatcgtggt gattgatgaa actgctgctg tcggctttaa cctctcttta ggcattggtt 1680 tcgaagcggg caacaagccg aaagaactgt acagcgaaga ggcagtcaac ggggaaactc 1740 agcaagcgca cttacaggcg attaaagagc tgatagcgcg tgacaaaaac cacccaagcg 1800 tggtgatgtg gagtattgcc aacgaaccgg atacccgtcc gcaaggtgca cgggaatatt 1860 tcgcgccact ggcggaagca acgcgtaaac tcgacccgac gcgtccgatc acctgcgtca 1920 atgtaatgtt ctgcgacgct cacaccgata ccatcagcga tctctttgat gtgctgtgcc 1980 tgaaccgtta ttacggatgg tatgtccaaa gcggcgattt ggaaacggca gagaaggtac 2040 tggaaaaaga acttctggcc tggcaggaga aactgcatca gccgattatc atcaccgaat 2100 acggcgtgga tacgttagcc gggctgcact caatgtacac cgacatgtgg agtgaagagt 2160 atcagtgtgc atggctggat atgtatcacc gcgtctttga tcgcgtcagc gccgtcgtcg 2220 gtgaacaggt atggaatttc gccgattttg cgacctcgca aggcatattg cgcgttggcg 2280 gtaacaagaa agggatcttc actcgcgacc gcaaaccgaa gtcggcggct tttctgctgc 2340 aaaaacgctg gactggcatg aacttcggtg aaaaaccgca gcagggaggc aaacaatgaa 2400 tcaacaactc tcctggcgca ccatcgtcgg ctacagcctc gggaattaga tccccgaatt 2460 tccccgatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc 2520 ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt 2580 aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt 2640 aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt 2700 catctatgtt actagatcgg gaatt 2725 44 9172 DNA Artificial artificial sequence vector pNOV2117 44 aagcttggcg cgccggtacc agcttgcatg cctgcagtgc agcgtgaccc ggtcgtgccc 60 ctctctagag ataatgagca ttgcatgtct aagttataaa aaattaccac atattttttt 120 tgtcacactt gtttgaagtg cagtttatct atctttatac atatatttaa actttactct 180 acgaataata taatctatag tactacaata atatcagtgt tttagagaat catataaatg 240 aacagttaga catggtctaa aggacaattg agtattttga caacaggact ctacagtttt 300 atctttttag tgtgcatgtg ttctcctttt tttttgcaaa tagcttcacc tatataatac 360 ttcatccatt ttattagtac atccatttag ggtttagggt taatggtttt tatagactaa 420 tttttttagt acatctattt tattctattt tagcctctaa attaagaaaa ctaaaactct 480 attttagttt ttttatttaa taatttagat ataaaataga ataaaataaa gtgactaaaa 540 attaaacaaa taccctttaa gaaattaaaa aaactaagga aacatttttc ttgtttcgag 600 tagataatgc cagcctgtta aacgccgtcg acgagtctaa cggacaccaa ccagcgaacc 660 agcagcgtcg cgtcgggcca agcgaagcag acggcacggc atctctgtcg ctgcctctgg 720 acccctctcg agagttccgc tccaccgttg gacttgctcc gctgtcggca tccagaaatt 780 gcgtggcgga gcggcagacg tgagccggca cggcaggcgg cctcctcctc ctctcacggc 840 accggcagct acgggggatt cctttcccac cgctccttcg ctttcccttc ctcgcccgcc 900 gtaataaata gacaccccct ccacaccctc tttccccaac ctcgtgttgt tcggagcgca 960 cacacacaca accagatctc ccccaaatcc acccgtcggc acctccgctt caaggtacgc 1020 cgctcgtcct cccccccccc ccctctctac cttctctaga tcggcgttcc ggtccatggt 1080 tagggcccgg tagttctact tctgttcatg tttgtgttag atccgtgttt gtgttagatc 1140 cgtgctgcta gcgttcgtac acggatgcga cctgtacgtc agacacgttc tgattgctaa 1200 cttgccagtg tttctctttg gggaatcctg ggatggctct agccgttccg cagacgggat 1260 cgatttcatg attttttttg tttcgttgca tagggtttgg tttgcccttt tcctttattt 1320 caatatatgc cgtgcacttg tttgtcgggt catcttttca tgcttttttt tgtcttggtt 1380 gtgatgatgt ggtctggttg ggcggtcgtt ctagatcgga gtagaattct gtttcaaact 1440 acctggtgga tttattaatt ttggatctgt atgtgtgtgc catacatatt catagttacg 1500 aattgaagat gatggatgga aatatcgatc taggataggt atacatgttg atgcgggttt 1560 tactgatgca tatacagaga tgctttttgt tcgcttggtt gtgatgatgt ggtgtggttg 1620 ggcggtcgtt cattcgttct agatcggagt agaatactgt ttcaaactac ctggtgtatt 1680 tattaatttt ggaactgtat gtgtgtgtca tacatcttca tagttacgag tttaagatgg 1740 atggaaatat cgatctagga taggtataca tgttgatgtg ggttttactg atgcatatac 1800 atgatggcat atgcagcatc tattcatatg ctctaacctt gagtacctat ctattataat 1860 aaacaagtat gttttataat tattttgatc ttgatatact tggatgatgg catatgcagc 1920 agctatatgt ggattttttt agccctgcct tcatacgcta tttatttgct tggtactgtt 1980 tcttttgtcg atgctcaccc tgttgtttgg tgttacttct gcagggatcc ccgatcatgc 2040 aaaaactcat taactcagtg caaaactatg cctggggcag caaaacggcg ttgactgaac 2100 tttatggtat ggaaaatccg tccagccagc cgatggccga gctgtggatg ggcgcacatc 2160 cgaaaagcag ttcacgagtg cagaatgccg ccggagatat cgtttcactg cgtgatgtga 2220 ttgagagtga taaatcgact ctgctcggag aggccgttgc caaacgcttt ggcgaactgc 2280 ctttcctgtt caaagtatta tgcgcagcac agccactctc cattcaggtt catccaaaca 2340 aacacaattc tgaaatcggt tttgccaaag aaaatgccgc aggtatcccg atggatgccg 2400 ccgagcgtaa ctataaagat cctaaccaca agccggagct ggtttttgcg ctgacgcctt 2460 tccttgcgat gaacgcgttt cgtgaatttt ccgagattgt ctccctactc cagccggtcg 2520 caggtgcaca tccggcgatt gctcactttt tacaacagcc tgatgccgaa cgtttaagcg 2580 aactgttcgc cagcctgttg aatatgcagg gtgaagaaaa atcccgcgcg ctggcgattt 2640 taaaatcggc cctcgatagc cagcagggtg aaccgtggca aacgattcgt ttaatttctg 2700 aattttaccc ggaagacagc ggtctgttct ccccgctatt gctgaatgtg gtgaaattga 2760 accctggcga agcgatgttc ctgttcgctg aaacaccgca cgcttacctg caaggcgtgg 2820 cgctggaagt gatggcaaac tccgataacg tgctgcgtgc gggtctgacg cctaaataca 2880 ttgatattcc ggaactggtt gccaatgtga aattcgaagc caaaccggct aaccagttgt 2940 tgacccagcc ggtgaaacaa ggtgcagaac tggacttccc gattccagtg gatgattttg 3000 ccttctcgct gcatgacctt agtgataaag aaaccaccat tagccagcag agtgccgcca 3060 ttttgttctg cgtcgaaggc gatgcaacgt tgtggaaagg ttctcagcag ttacagctta 3120 aaccgggtga atcagcgttt attgccgcca acgaatcacc ggtgactgtc aaaggccacg 3180 gccgtttagc gcgtgtttac aacaagctgt aagagcttac tgaaaaaatt aacatctctt 3240 gctaagctgg gagctcgatc cgtcgacctg cagatcgttc aaacatttgg caataaagtt 3300 tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt ctgttgaatt 3360 acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga tgggttttta 3420 tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata tagcgcgcaa 3480 actaggataa attatcgcgc gcggtgtcat ctatgttact agatctgcta gccctgcagg 3540 aaatttaccg gtgcccgggc ggccagcatg gccgtatccg caatgtgtta ttaagttgtc 3600 taagcgtcaa tttgtttaca ccacaatata tcctgccacc agccagccaa cagctccccg 3660 accggcagct cggcacaaaa tcaccactcg atacaggcag cccatcagaa ttaattctca 3720 tgtttgacag cttatcatcg actgcacggt gcaccaatgc ttctggcgtc aggcagccat 3780 cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg 3840 cgcactcccg ttctggataa tgttttttgc gccgacatca taacggttct ggcaaatatt 3900 ctgaaatgag ctgttgacaa ttaatcatcc ggctcgtata atgtgtggaa ttgtgagcgg 3960 ataacaattt cacacaggaa acagaccatg agggaagcgt tgatcgccga agtatcgact 4020 caactatcag aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt gctggccgta 4080 catttgtacg gctccgcagt ggatggcggc ctgaagccac acagtgatat tgatttgctg 4140 gttacggtga ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg 4200 gaaacttcgg cttcccctgg agagagcgag attctccgcg ctgtagaagt caccattgtt 4260 gtgcacgacg acatcattcc gtggcgttat ccagctaagc gcgaactgca atttggagaa 4320 tggcagcgca atgacattct tgcaggtatc ttcgagccag ccacgatcga cattgatctg 4380 gctatcttgc tgacaaaagc aagagaacat agcgttgcct tggtaggtcc agcggcggag 4440 gaactctttg atccggttcc tgaacaggat ctatttgagg cgctaaatga aaccttaacg 4500 ctatggaact cgccgcccga ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc 4560 cgcatttggt acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg 4620 gcaatggagc gcctgccggc ccagtatcag cccgtcatac ttgaagctag gcaggcttat 4680 cttggacaag aagatcgctt ggcctcgcgc gcagatcagt tggaagaatt tgttcactac 4740 gtgaaaggcg agatcaccaa agtagtcggc aaataaagct ctagtggatc tccgtacccc 4800 cgggggatct ggctcgcggc ggacgcacga cgccggggcg agaccatagg cgatctccta 4860 aatcaatagt agctgtaacc tcgaagcgtt tcacttgtaa caacgattga gaatttttgt 4920 cataaaattg aaatacttgg ttcgcatttt tgtcatccgc ggtcagccgc aattctgacg 4980 aactgcccat ttagctggag atgattgtac atccttcacg tgaaaatttc tcaagcgctg 5040 tgaacaaggg ttcagatttt agattgaaag gtgagccgtt gaaacacgtt cttcttgtcg 5100 atgacgacgt cgctatgcgg catcttatta ttgaatacct tacgatccac gccttcaaag 5160 tgaccgcggt agccgacagc acccagttca caagagtact ctcttccgcg acggtcgatg 5220 tcgtggttgt tgatctaaat ttaggtcgtg aagatgggct cgagatcgtt cgtaatctgg 5280 cggcaaagtc tgatattcca atcataatta tcagtggcga ccgccttgag gagacggata 5340 aagttgttgc actcgagcta ggagcaagtg attttatcgc taagccgttc agtatcagag 5400 agtttctagc acgcattcgg gttgccttgc gcgtgcgccc caacgttgtc cgctccaaag 5460 accgacggtc tttttgtttt actgactgga cacttaatct caggcaacgt cgcttgatgt 5520 ccgaagctgg cggtgaggtg aaacttacgg caggtgagtt caatcttctc ctcgcgtttt 5580 tagagaaacc ccgcgacgtt ctatcgcgcg agcaacttct cattgccagt cgagtacgcg 5640 acgaggaggt ttatgacagg agtatagatg ttctcatttt gaggctgcgc cgcaaacttg 5700 aggcagatcc gtcaagccct caactgataa aaacagcaag aggtgccggt tatttctttg 5760 acgcggacgt gcaggtttcg cacgggggga cgatggcagc ctgagccaat tcccagatcc 5820 ccgaggaatc ggcgtgagcg gtcgcaaacc atccggcccg gtacaaatcg gcgcggcgct 5880 gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc aacgcatcga 5940 ggcagaagca cgccccggtg aatcgtggca agcggccgct gatcgaatcc gcaaagaatc 6000 ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg gcgacgagca 6060 accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc gcagcatcat 6120 ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg tgatccgcta 6180 cgagcttcca gacgggcacg tagaggtttc cgcagggccg gccggcatgg ccagtgtgtg 6240 ggattacgac ctggtactga tggcggtttc ccatctaacc gaatccatga accgataccg 6300 ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg acgtactcaa 6360 gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa cctgcattcg 6420 gttaaacacc acgcacgttg ccatgcagcg tacgaagaag gccaagaacg gccgcctggt 6480 gacggtatcc gagggtgaag ccttgattag ccgctacaag atcgtaaaga gcgaaaccgg 6540 gcggccggag tacatcgaga tcgagctagc tgattggatg taccgcgaga tcacagaagg 6600 caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc ccggcatcgg 6660 ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca gatggttgtt 6720 caagacgatc tacgaacgca gtggcagcgc cggagagttc aagaagttct gtttcaccgt 6780 gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg aggcggggca 6840 ggctggcccg atcctagtca tgcgctaccg caacctgatc gagggcgaag catccgccgg 6900 ttcctaatgt acggagcaga tgctagggca aattgcccta gcaggggaaa aaggtcgaaa 6960 aggtctcttt cctgtggata gcacgtacat tgggaaccca aagccgtaca ttgggaaccg 7020 gaacccgtac attgggaacc caaagccgta cattgggaac cggtcacaca tgtaagtgac 7080 tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac ttattaaaac 7140 tcttaaaacc cgcctggcct gtgcataact gtctggccag cgcacagccg aagagctgca 7200 aaaagcgcct acccttcggt cgctgcgctc cctacgcccc gccgcttcgc gtcggcctat 7260 cgcggccgct ggccgctcaa aaatggctgg cctacggcca ggcaatctac cagggcgcgg 7320 acaagccgcg ccgtcgccac tcgaccgccg gcgctgaggt ctgcctcgtg aagaaggtgt 7380 tgctgactca taccaggcct gaatcgcccc atcatccagc cagaaagtga gggagccacg 7440 gttgatgaga gctttgttgt aggtggacca gttggtgatt ttgaactttt gctttgccac 7500 ggaacggtct gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg 7560 atttattcaa caaagccgcc gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac 7620 caattaacca attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc 7680 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac 7740 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt 7800 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa 7860 tcaccatgag tgacgactga atccggtgag aatggcaaaa gctctgcatt aatgaatcgg 7920 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 7980 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 8040 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 8100 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 8160 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 8220 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 8280 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 8340 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 8400 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 8460 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 8520 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 8580 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 8640 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 8700 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 8760 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 8820 cttcacctag atccttttga tccggaatta attcctgtgg ttggcatgca catacaaatg 8880 gacgaacgga taaacctttt cacgcccttt taaatatccg attattctaa taaacgctct 8940 tttctcttag gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc 9000 gggaaacgac aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg 9060 atgacgcggg acaagccgtt ttacgtttgg aactgacaga accgcaacgc tgcaggaatt 9120 ggccgcagcg gccatttaaa tcaattgggc gcgccgaatt cgagctcggt ac 9172 45 8849 DNA Artificial artificial sequence vector pNOV4200 45 ggtacccccg ggggatcctc tagagtcgac catggtgatc actgcaggca tgcaagcttc 60 gtacgttaat taattcgaat ccggagcggc cgcacgcgtg ggcccgttta aacctcgaga 120 gatctgctag ccctgcagga aatttaccgg tgcccgtacc ggatttggag ccaagtctca 180 taaacgccat tgtggaagaa agtcttgagt tggtggtaat gtaacagagt agtaagaaca 240 gagaagagag agagtgtgag atacatgaat tgtcgggcaa caaaaatcct gaacatctta 300 ttttagcaaa gagaaagagt tccgagtctg tagcagaaga gtgaggagaa atttaagctc 360 ttggacttgt gaattgttcc gcctcttgaa tacttcttca atcctcatat attcttcttc 420 tatgttacct gaaaaccggc atttaatctc gcgggtttat tccggttcaa catttttttt 480 gttttgagtt attatctggg cttaataacg caggcctgaa ataaattcaa ggcccaactg 540 tttttttttt taagaagttg ctgttaaaaa aaaaaaaagg gaattaacaa caacaacaaa 600 aaaagataaa gaaaataata acaattactt taattgtaga ctaaaaaaac atagatttta 660 tcatgaaaaa aagagaaaag aaataaaaac ttggatcaaa aaaaaacata cagatcttct 720 aattattaac ttttcttaaa aattaggtcc tttttcccaa caattaggtt tagagttttg 780 gaattaaacc aaaaagattg ttctaaaaaa tactcaaatt tggtagataa gtttccttat 840 tttaattagt caatggtaga tacttttttt tcttttcttt attagagtag attagaatct 900 tttatgccaa gtattgataa attaaatcaa gaagataaac tatcataatc aacatgaaat 960 taaaagaaaa atctcatata tagtattagt attctctata tatattatga ttgcttattc 1020 ttaatgggtt gggttaacca agacatagtc ttaatggaaa gaatcttttt tgaacttttt 1080 ccttattgat taaattcttc tatagaaaag aaagaaatta tttgaggaaa agtatataca 1140 aaaagaaaaa tagaaaaatg tcagtgaagc agatgtaatg gatgacctaa tccaaccacc 1200 accataggat gtttctactt gagtcggtct tttaaaaacg cacggtggaa aatatgacac 1260 gtatcatatg attccttcct ttagtttcgt gataataatc ctcaactgat atcttccttt 1320 ttttgttttg gctaaagata ttttattctc attaatagaa aagacggttt tgggcttttg 1380 gtttgcgata taaagaagac cttcgtgtgg aagataataa ttcatccttt cgtctttttc 1440 tgactcttca atctctccca aagcctaaag cgatctctgc aaatctctcg cgactctctc 1500 tttcaaggta tattttctga ttctttttgt ttttgattcg tatctgatct ccaatttttg 1560 ttatgtggat tattgaatct tttgtataaa ttgcttttga caatattgtt cgtttcgtca 1620 atccagcttc taaattttgt cctgattact aagatatcga ttcgtagtgt ttacatctgt 1680 gtaatttctt gcttgattgt gaaattagga ttttcaagga cgatctattc aatttttgtg 1740 ttttctttgt tcgattctct ctgttttagg tttcttatgt ttagatccgt ttctctttgg 1800 tgttgttttg atttctctta cggcttttga tttggtatat gttcgctgat tggtttctac 1860 ttgttctatt gttttatttc aggtggatct cgactctagg ggggcaataa gatatgaaaa 1920 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct 1980 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag 2040 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg 2100 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggcat 2160 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc 2220 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg 2280 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc 2340 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc 2400 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc 2460 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca 2520 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg 2580 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg 2640 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc 2700 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt 2760 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga 2820 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag 2880 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaataga 2940 gtagatgccg accgggatcc ccgaatttcc ccgatcgttc aaacatttgg caataaagtt 3000 tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt ctgttgaatt 3060 acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga tgggttttta 3120 tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata tagcgcgcaa 3180 actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgggaa ttgggtacgg 3240 gcggccagca tggccgtatc cgcaatgtgt tattaagttg tctaagcgtc aatttgttta 3300 caccacaata tatcctgcca ccagccagcc aacagctccc cgaccggcag ctcggcacaa 3360 aatcaccact cgatacaggc agcccatcag aattaattct catgtttgac agcttatcat 3420 cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc atcggaagct gtggtatggc 3480 tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc cgttctggat 3540 aatgtttttt gcgccgacat cataacggtt ctggcaaata ttctgaaatg agctgttgac 3600 aattaatcat ccggctcgta taatgtgtgg aattgtgagc ggataacaat ttcacacagg 3660 aaacagacca tgagggaagc gttgatcgcc gaagtatcga ctcaactatc agaggtagtt 3720 ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg tacatttgta cggctccgca 3780 gtggatggcg gcctgaagcc acacagtgat attgatttgc tggttacggt gaccgtaagg 3840 cttgatgaaa caacgcggcg agctttgatc aacgaccttt tggaaacttc ggcttcccct 3900 ggagagagcg agattctccg cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt 3960 ccgtggcgtt atccagctaa gcgcgaactg caatttggag aatggcagcg caatgacatt 4020 cttgcaggta tcttcgagcc agccacgatc gacattgatc tggctatctt gctgacaaaa 4080 gcaagagaac atagcgttgc cttggtaggt ccagcggcgg aggaactctt tgatccggtt 4140 cctgaacagg atctatttga ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc 4200 gactgggctg gcgatgagcg aaatgtagtg cttacgttgt cccgcatttg gtacagcgca 4260 gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact gggcaatgga gcgcctgccg 4320 gcccagtatc agcccgtcat acttgaagct aggcaggctt atcttggaca agaagatcgc 4380 ttggcctcgc gcgcagatca gttggaagaa tttgttcact acgtgaaagg cgagatcacc 4440 aaagtagtcg gcaaataaag ctctagtgga tctccgtacc cccgggggat ctggctcgcg 4500 gcggacgcac gacgccgggg cgagaccata ggcgatctcc taaatcaata gtagctgtaa 4560 cctcgaagcg tttcacttgt aacaacgatt gagaattttt gtcataaaat tgaaatactt 4620 ggttcgcatt tttgtcatcc gcggtcagcc gcaattctga cgaactgccc atttagctgg 4680 agatgattgt acatccttca cgtgaaaatt tctcaagcgc tgtgaacaag ggttcagatt 4740 ttagattgaa aggtgagccg ttgaaacacg ttcttcttgt cgatgacgac gtcgctatgc 4800 ggcatcttat tattgaatac cttacgatcc acgccttcaa agtgaccgcg gtagccgaca 4860 gcacccagtt cacaagagta ctctcttccg cgacggtcga tgtcgtggtt gttgatctag 4920 atttaggtcg tgaagatggg ctcgagatcg ttcgtaatct ggcggcaaag tctgatattc 4980 caatcataat tatcagtggc gaccgccttg aggagacgga taaagttgtt gcactcgagc 5040 taggagcaag tgattttatc gctaagccgt tcagtatcag agagtttcta gcacgcattc 5100 gggttgcctt gcgcgtgcgc cccaacgttg tccgctccaa agaccgacgg tctttttgtt 5160 ttactgactg gacacttaat ctcaggcaac gtcgcttgat gtccgaagct ggcggtgagg 5220 tgaaacttac ggcaggtgag ttcaatcttc tcctcgcgtt tttagagaaa ccccgcgacg 5280 ttctatcgcg cgagcaactt ctcattgcca gtcgagtacg cgacgaggag gtttatgaca 5340 ggagtataga tgttctcatt ttgaggctgc gccgcaaact tgaggcagat ccgtcaagcc 5400 ctcaactgat aaaaacagca agaggtgccg gttatttctt tgacgcggac gtgcaggttt 5460 cgcacggggg gacgatggca gcctgagcca attcccagat ccccgaggaa tcggcgtgag 5520 cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga 5580 gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg 5640 tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 5700 cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc 5760 gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg 5820 tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca 5880 cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact 5940 gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa 6000 gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga 6060 tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt 6120 tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga 6180 agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga 6240 gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 6300 gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct 6360 ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg 6420 cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc 6480 aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt 6540 catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca 6600 gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga 6660 tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa 6720 cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa 6780 aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc 6840 ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg 6900 gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc 6960 aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc 7020 actcgaccgc cggcgctgag gtctgcctcg tgaagaaggt gttgctgact cataccaggc 7080 ctgaatcgcc ccatcatcca gccagaaagt gagggagcca cggttgatga gagctttgtt 7140 gtaggtggac cagttggtga ttttgaactt ttgctttgcc acggaacggt ctgcgttgtc 7200 gggaagatgc gtgatctgat ccttcaactc agcaaaagtt cgatttattc aacaaagccg 7260 ccgtcccgtc aagtcagcgt aatgctctgc cagtgttaca accaattaac caattctgat 7320 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata 7380 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat 7440 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct 7500 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact 7560 gaatccggtg agaatggcaa aagctctgca ttaatgaatc ggccaacgcg cggggagagg 7620 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 7680 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 7740 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 7800 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 7860 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 7920 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 7980 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 8040 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 8100 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 8160 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 8220 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 8280 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 8340 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 8400 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 8460 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 8520 gatccggaat taattcctgt ggttggcatg cacatacaaa tggacgaacg gataaacctt 8580 ttcacgccct tttaaatatc cgattattct aataaacgct cttttctctt aggtttaccc 8640 gccaatatat cctgtcaaac actgatagtt taaactgaag gcgggaaacg acaatctgat 8700 catgagcgga gaattaaggg agtcacgtta tgacccccgc cgatgacgcg ggacaagccg 8760 ttttacgttt ggaactgaca gaaccgcaac gctgcaggaa ttggccgcag cggccattta 8820 aatcaattgg gcgcgccgaa ttcgagctc 8849 46 2949 DNA Artificial artificial sequence ZmUbi-GFP-35S term expression cassette 46 gcatgcctgc agtgcagcgt gacccggtcg tgcccctctc tagagataat gagcattgca 60 tgtctaagtt ataaaaaatt accacatatt ttttttgtca cacttgtttg aagtgcagtt 120 tatctatctt tatacatata tttaaacttt actctacgaa taatataatc tatagtacta 180 caataatatc agtgttttag agaatcatat aaatgaacag ttagacatgg tctaaaggac 240 aattgagtat tttgacaaca ggactctaca gttttatctt tttagtgtgc atgtgttctc 300 cttttttttt gcaaatagct tcacctatat aatacttcat ccattttatt agtacatcca 360 tttagggttt agggttaatg gtttttatag actaattttt ttagtacatc tattttattc 420 tattttagcc tctaaattaa gaaaactaaa actctatttt agttttttta tttaataatt 480 tagatataaa atagaataaa ataaagtgac taaaaattaa acaaataccc tttaagaaat 540 taaaaaaact aaggaaacat ttttcttgtt tcgagtagat aatgccagcc tgttaaacgc 600 cgtcgacgag tctaacggac accaaccagc gaaccagcag cgtcgcgtcg ggccaagcga 660 agcagacggc acggcatctc tgtcgctgcc tctggacccc tctcgagagt tccgctccac 720 cgttggactt gctccgctgt cggcatccag aaattgcgtg gcggagcggc agacgtgagc 780 cggcacggca ggcggcctcc tcctcctctc acggcacggc agctacgggg gattcctttc 840 ccaccgctcc ttcgctttcc cttcctcgcc cgccgtaata aatagacacc ccctccacac 900 cctctttccc caacctcgtg ttgttcggag cgcacacaca cacaaccaga tctcccccaa 960 atccacccgt cggcacctcc gcttcaaggt acgccgctcg tcctcccccc ccccccctct 1020 ctaccttctc tagatcggcg ttccggtcca tggttagggc ccggtagttc tacttctgtt 1080 catgtttgtg ttagatccgt gtttgtgtta gatccgtgct gctagcgttc gtacacggat 1140 gcgacctgta cgtcagacac gttctgattg ctaacttgcc agtgtttctc tttggggaat 1200 cctgggatgg ctctagccgt tccgcagacg ggatcgattt catgattttt tttgtttcgt 1260 tgcatagggt ttggtttgcc cttttccttt atttcaatat atgccgtgca cttgtttgtc 1320 gggtcatctt ttcatgcttt tttttgtctt ggttgtgatg atgtggtctg gttgggcggt 1380 cgttctagat cggagtagaa ttctgtttca aactacctgg tggatttatt aattttggat 1440 ctgtatgtgt gtgccataca tattcatagt tacgaattga agatgatgga tggaaatatc 1500 gatctaggat aggtatacat gttgatgcgg gttttactga tgcatataca gagatgcttt 1560 ttgttcgctt ggttgtgatg atgtggtgtg gttgggcggt cgttcattcg ttctagatcg 1620 gagtagaata ctgtttcaaa ctacctggtg tatttattaa ttttggaact gtatgtgtgt 1680 gtcatacatc ttcatagtta cgagtttaag atggatggaa atatcgatct aggataggta 1740 tacatgttga tgtgggtttt actgatgcat atacatgatg gcatatgcag catctattca 1800 tatgctctaa ccttgagtac ctatctatta taataaacaa gtatgtttta taattatttt 1860 gatcttgata tacttggatg atggcatatg cagcagctat atgtggattt ttttagccct 1920 gccttcatac gctatttatt tgcttggtac tgtttctttt gtcgatgctc accctgttgt 1980 ttggtgttac ttctgcaggt cgactctaga ggatccacca tgctgcagat gagcaagggc 2040 gaggagctgt tcaccggcgt ggtgccaatc ctggtggagc tggacggcga cgtgaacggc 2100 cacaagttca gcgtgagcgg cgagggcgag ggcgacgcga cctacggcaa gctgaccctg 2160 aagttcatct gtaccaccgg caagctcccg gtcccgtggc cgaccctggt gaccaccttc 2220 acctacggcg tgcagtgttt cagccgctac ccggaccaca tgaagcgcca cgacttcttc 2280 aagagcgcca tgccggaggg ctacgtgcag gagcgcacca tcagcttcaa ggacgacggc 2340 aactacaaga cccgcgccga ggtgaagttc gagggcgaca cactagtgaa ccgcatcgag 2400 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg gccacaagct ggagtacaac 2460 tacaacagcc acaacgtgta catcaccgcg gacaagcaga agaacggcat caaggcgaac 2520 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tggccgacca ctaccagcag 2580 aacaccccga tcggcgacgg tcctgtgctg ctgccggaca accactacct gagcacccag 2640 agcgccctga gcaaggaccc gaacgagaag cgcgaccaca tggtgctgct ggagttcgtg 2700 accgccgccg gcatcaccca cggcatggac gagctgtaca aggttaacta gagctcaaga 2760 tctgttctgc acaaagtgga gtagtcagtc atcgatcagg aaccagacac cagactttta 2820 ttcatacagt gaagtgaagt gaagtgcagt gcagtgagtt gctggttttt gtacaactta 2880 gtatgtattt gtatttgtaa aatacttcta tcaataaaat ttctaattcc taaaaccaaa 2940 atccagtgg 2949 47 4341 DNA Artificial artificial sequence ZmUBi-GIG-nos expression cassette 47 cctgcagtgc agcgtgaccc ggtcgtgccc ctctctagag ataatgagca ttgcatgtct 60 aagttataaa aaattaccac atattttttt tgtcacactt gtttgaagtg cagtttatct 120 atctttatac atatatttaa actttactct acgaataata taatctatag tactacaata 180 atatcagtgt tttagagaat catataaatg aacagttaga catggtctaa aggacaattg 240 agtattttga caacaggact ctacagtttt atctttttag tgtgcatgtg ttctcctttt 300 tttttgcaaa tagcttcacc tatataatac ttcatccatt ttattagtac atccatttag 360 ggtttagggt taatggtttt tatagactaa tttttttagt acatctattt tattctattt 420 tagcctctaa attaagaaaa ctaaaactct attttagttt ttttatttaa taatttagat 480 ataaaataga ataaaataaa gtgactaaaa attaaacaaa taccctttaa gaaattaaaa 540 aaactaagga aacatttttc ttgtttcgag tagataatgc cagcctgtta aacgccgtcg 600 acgagtctaa cggacaccaa ccagcgaacc agcagcgtcg cgtcgggcca agcgaagcag 660 acggcacggc atctctgtcg ctgcctctgg acccctctcg agagttccgc tccaccgttg 720 gacttgctcc gctgtcggca tccagaaatt gcgtggcgga gcggcagacg tgagccggca 780 cggcaggcgg cctcctcctc ctctcacggc accggcagct acgggggatt cctttcccac 840 cgctccttcg ctttcccttc ctcgcccgcc gtaataaata gacaccccct ccacaccctc 900 tttccccaac ctcgtgttgt tcggagcgca cacacacaca accagatctc ccccaaatcc 960 acccgtcggc acctccgctt caaggtacgc cgctcgtcct cccccccccc ccctctctac 1020 cttctctaga tcggcgttcc ggtccatggt tagggcccgg tagttctact tctgttcatg 1080 tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac acggatgcga 1140 cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg gggaatcctg 1200 ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg tttcgttgca 1260 tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg tttgtcgggt 1320 catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg ggcggtcgtt 1380 ctagatcgga gtagaattct gtttcaaact acctggtgga tttattaatt ttggatctgt 1440 atgtgtgtgc catacatatt catagttacg aattgaagat gatggatgga aatatcgatc 1500 taggataggt atacatgttg atgcgggttt tactgatgca tatacagaga tgctttttgt 1560 tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct agatcggagt 1620 agaatactgt ttcaaactac ctggtgtatt tattaatttt ggaactgtat gtgtgtgtca 1680 tacatcttca tagttacgag tttaagatgg atggaaatat cgatctagga taggtataca 1740 tgttgatgtg ggttttactg atgcatatac atgatggcat atgcagcatc tattcatatg 1800 ctctaacctt gagtacctat ctattataat aaacaagtat gttttataat tattttgatc 1860 ttgatatact tggatgatgg catatgcagc agctatatgt ggattttttt agccctgcct 1920 tcatacgcta tttatttgct tggtactgtt tcttttgtcg atgctcaccc tgttgtttgg 1980 tgttacttct gcagggatcc cctcgaggtc gaccatggtc cgtcctgtag aaaccccaac 2040 ccgtgaaatc aaaaaactcg acggcctgtg ggcattcagt ctggatcgcg aaaactgtgg 2100 aattgatcag cgttggtggg aaagcgcgtt acaagaaagc cgggcaattg ctgtgccagg 2160 cagttttaac gatcagttcg ccgatgcaga tattcgtaat tatgcgggca acgtctggta 2220 tcagcgcgaa gtctttatac cgaaaggttg ggcaggccag cgtatcgtgc tgcgtttcga 2280 tgcggtcact cattacggca aagtgtgggt caataatcag gaagtgatgg agcatcaggg 2340 cggctatacg ccatttgaag ccgatgtcac gccgtatgtt attgccggga aaagtgtacg 2400 taagtttctg cttctacctt tgatatatat ataataatta tcattaatta gtagtaatat 2460 aatatttcaa atattttttt caaaataaaa gaatgtagta tatagcaatt gcttttctgt 2520 agtttataag tgtgtatatt ttaatttata acttttctaa tatatgacca aaatttgttg 2580 atgtgcaggt atcaccgttt gtgtgaacaa cgaactgaac tggcagacta tcccgccggg 2640 aatggtgatt accgacgaaa acggcaagaa aaagcagtct tacttccatg atttctttaa 2700 ctatgccgga atccatcgca gcgtaatgct ctacaccacg ccgaacacct gggtggacga 2760 tatcaccgtg gtgacgcatg tcgcgcaaga ctgtaaccac gcgtctgttg actggcaggt 2820 ggtggccaat ggtgatgtca gcgttgaact gcgtgatgcg gatcaacagg tggttgcaac 2880 tggacaaggc actagcggga ctttgcaagt ggtgaatccg cacctctggc aaccgggtga 2940 aggttatctc tatgaactgt gcgtcacagc caaaagccag acagagtgtg atatctaccc 3000 gcttcgcgtc ggcatccggt cagtggcagt gaagggcgaa cagttcctga ttaaccacaa 3060 accgttctac tttactggct ttggtcgtca tgaagatgcg gacttgcgtg gcaaaggatt 3120 cgataacgtg ctgatggtgc acgaccacgc attaatggac tggattgggg ccaactccta 3180 ccgtacctcg cattaccctt acgctgaaga gatgctcgac tgggcagatg aacatggcat 3240 cgtggtgatt gatgaaactg ctgctgtcgg ctttaacctc tctttaggca ttggtttcga 3300 agcgggcaac aagccgaaag aactgtacag cgaagaggca gtcaacgggg aaactcagca 3360 agcgcactta caggcgatta aagagctgat agcgcgtgac aaaaaccacc caagcgtggt 3420 gatgtggagt attgccaacg aaccggatac ccgtccgcaa ggtgcacggg aatatttcgc 3480 gccactggcg gaagcaacgc gtaaactcga cccgacgcgt ccgatcacct gcgtcaatgt 3540 aatgttctgc gacgctcaca ccgataccat cagcgatctc tttgatgtgc tgtgcctgaa 3600 ccgttattac ggatggtatg tccaaagcgg cgatttggaa acggcagaga aggtactgga 3660 aaaagaactt ctggcctggc aggagaaact gcatcagccg attatcatca ccgaatacgg 3720 cgtggatacg ttagccgggc tgcactcaat gtacaccgac atgtggagtg aagagtatca 3780 gtgtgcatgg ctggatatgt atcaccgcgt ctttgatcgc gtcagcgccg tcgtcggtga 3840 acaggtatgg aatttcgccg attttgcgac ctcgcaaggc atattgcgcg ttggcggtaa 3900 caagaaaggg atcttcactc gcgaccgcaa accgaagtcg gcggcttttc tgctgcaaaa 3960 acgctggact ggcatgaact tcggtgaaaa accgcagcag ggaggcaaac aatgaatcaa 4020 caactctcct ggcgcaccat cgtcggctac agcctcggga attagatccc cgaatttccc 4080 cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 4140 gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 4200 catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 4260 cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 4320 tatgttacta gatcgggaat t 4341 48 2943 DNA Artificial artificial sequence Ubq3(At)-synGFPI-nos expression cassette 48 accggatttg gagccaagtc tcataaacgc cattgtggaa gaaagtcttg agttggtggt 60 aatgtaacag agtagtaaga acagagaaga gagagagtgt gagatacatg aattgtcggg 120 caacaaaaat cctgaacatc ttattttagc aaagagaaag agttccgagt ctgtagcaga 180 agagtgagga gaaatttaag ctcttggact tgtgaattgt tccgcctctt gaatacttct 240 tcaatcctca tatattcttc ttctatgtta cctgaaaacc ggcatttaat ctcgcgggtt 300 tattccggtt caacattttt tttgttttga gttattatct gggcttaata acgcaggcct 360 gaaataaatt caaggcccaa ctgttttttt ttttaagaag ttgctgttaa aaaaaaaaaa 420 agggaattaa caacaacaac aaaaaaagat aaagaaaata ataacaatta ctttaattgt 480 agactaaaaa aacatagatt ttatcatgaa aaaaagagaa aagaaataaa aacttggatc 540 aaaaaaaaaa catacagatc ttctaattat taacttttct taaaaattag gtcctttttc 600 ccaacaatta ggtttagagt tttggaatta aaccaaaaag attgttctaa aaaatactca 660 aatttggtag ataagtttcc ttattttaat tagtcaatgg tagatacttt tttttctttt 720 ctttattaga gtagattaga atcttttatg ccaagtattg ataaattaaa tcaagaagat 780 aaactatcat aatcaacatg aaattaaaag aaaaatctca tatatagtat tagtattctc 840 tatatatatt atgattgctt attcttaatg ggttgggtta accaagacat agtcttaatg 900 gaaagaatct tttttgaact ttttccttat tgattaaatt cttctataga aaagaaagaa 960 attatttgag gaaaagtata tacaaaaaga aaaatagaaa aatgtcagtg aagcagatgt 1020 aatggatgac ctaatccaac caccaccata ggatgtttct acttgagtcg gtcttttaaa 1080 aacgcacggt ggaaaatatg acacgtatca tatgattcct tcctttagtt tcgtgataat 1140 aatcctcaac tgatatcttc ctttttttgt tttggctaaa gatattttat tctcattaat 1200 agaaaagacg gttttgggct tttggtttgc gatataaaga agaccttcgt gtggaagata 1260 ataattcatc ctttcgtctt tttctgactc ttcaatctct cccaaagcct aaagcgatct 1320 ctgcaaatct ctcgcgactc tctctttcaa ggtatatttt ctgattcttt ttgtttttga 1380 ttcgtatctg atctccaatt tttgttatgt ggattattga atcttttgta taaattgctt 1440 ttgacaatat tgttcgtttc gtcaatccag cttctaaatt ttgtcctgat tactaagata 1500 tcgattcgta gtgtttacat ctgtgtaatt tcttgcttga ttgtgaaatt aggattttca 1560 aggacgatct attcaatttt tgtgttttct ttgttcgatt ctctctgttt taggtttctt 1620 atgtttagat ccgtttctct ttggtgttgt tttgatttct cttacggctt ttgatttggt 1680 atatgttcgc tgattggttt ctacttgttc tattgtttta tttcaggtgg atccaccatg 1740 ctgcagatga gcaagggcga ggagctgttc accggcgtgg tgccaatcct ggtggagctg 1800 gacggcgacg tgaacggcca caagttcagc gtgagcggcg agggcgaggg cgacgcgacc 1860 tacggcaagc tgaccctgaa gttcatctgt accaccggca agctcccggt cccgtggccg 1920 accctggtga ccaccttcac ctacggcgtg cagtgtttca gccgctaccc ggaccacatg 1980 aagcgccacg acttcttcaa gagcgccatg ccggagggct acgtaagttt ctgcttctac 2040 ctttgatata tatataataa ttatcattaa ttagtagtaa tataatattt caaatatttt 2100 tttcaaaata aaagaatgta gtatatagca attgcttttc tgtagtttat aagtgtgtat 2160 attttaattt ataacttttc taatatatga ccaaaatttg ttgatgtgca ggtgcaggag 2220 cgcaccatca gcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 2280 ggcgacacac tagtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 2340 atcctgggcc acaagctgga gtacaactac aacagccaca atgtgtacat caccgcggac 2400 aagcagaaga acggcatcaa ggcgaacttc aagatccgcc acaatatcga ggacggcagc 2460 gtgcagctgg ccgaccacta ccagcagaac accccgatcg gcgacggtcc tgtgctgctg 2520 ccggacaacc actacctgag cacccagagc gccctgagca aggacccgaa cgagaagcgc 2580 gaccacatgg tgctgctgga gttcgtgacc gccgccggca tcacccacgg catggacgag 2640 ctgtacaagg ttaactagag ctctagatcc ccgaatttcc ccgatcgttc aaacatttgg 2700 caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt 2760 ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga 2820 tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata 2880 tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgggaa 2940 ttg 2943 49 4072 DNA Artificial artificial sequence Ubq3(At)-GIG-nos expression cassette 49 cggatttgga gccaagtctc ataaacgcca ttgtggaaga aagtcttgag ttggtggtaa 60 tgtaacagag tagtaagaac agagaagaga gagagtgtga gatacatgaa ttgtcgggca 120 acaaaaatcc tgaacatctt attttagcaa agagaaagag ttccgagtct gtagcagaag 180 agtgaggaga aatttaagct cttggacttg tgaattgttc cgcctcttga atacttcttc 240 aatcctcata tattcttctt ctatgttacc tgaaaaccgg catttaatct cgcgggttta 300 ttccggttca acattttttt tgttttgagt tattatctgg gcttaataac gcaggcctga 360 aataaattca aggcccaact gttttttttt ttaagaagtt gctgttaaaa aaaaaaaaag 420 ggaattaaca acaacaacaa aaaaagataa agaaaataat aacaattact ttaattgtag 480 actaaaaaaa catagatttt atcatgaaaa aaagagaaaa gaaataaaaa cttggatcaa 540 aaaaaaacat acagatcttc taattattaa cttttcttaa aaattaggtc ctttttccca 600 acaattaggt ttagagtttt ggaattaaac caaaaagatt gttctaaaaa atactcaaat 660 ttggtagata agtttcctta ttttaattag tcaatggtag atactttttt ttcttttctt 720 tattagagta gattagaatc ttttatgcca agtattgata aattaaatca agaagataaa 780 ctatcataat caacatgaaa ttaaaagaaa aatctcatat atagtattag tattctctat 840 atatattatg attgcttatt cttaatgggt tgggttaacc aagacatagt cttaatggaa 900 agaatctttt ttgaactttt tccttattga ttaaattctt ctatagaaaa gaaagaaatt 960 atttgaggaa aagtatatac aaaaagaaaa atagaaaaat gtcagtgaag cagatgtaat 1020 ggatgaccta atccaaccac caccatagga tgtttctact tgagtcggtc ttttaaaaac 1080 gcacggtgga aaatatgaca cgtatcatat gattccttcc tttagtttcg tgataataat 1140 cctcaactga tatcttcctt tttttgtttt ggctaaagat attttattct cattaataga 1200 aaagacggtt ttgggctttt ggtttgcgat ataaagaaga ccttcgtgtg gaagataata 1260 attcatcctt tcgtcttttt ctgactcttc aatctctccc aaagcctaaa gcgatctctg 1320 caaatctctc gcgactctct ctttcaaggt atattttctg attctttttg tttttgattc 1380 gtatctgatc tccaattttt gttatgtgga ttattgaatc ttttgtataa attgcttttg 1440 acaatattgt tcgtttcgtc aatccagctt ctaaattttg tcctgattac taagatatcg 1500 attcgtagtg tttacatctg tgtaatttct tgcttgattg tgaaattagg attttcaagg 1560 acgatctatt caatttttgt gttttctttg ttcgattctc tctgttttag gtttcttatg 1620 tttagatccg tttctctttg gtgttgtttt gatttctctt acggcttttg atttggtata 1680 tgttcgctga ttggtttcta cttgttctat tgttttattt caggtggatc ccctcgaggt 1740 cgaccatggt ccgtcctgta gaaaccccaa cccgtgaaat caaaaaactc gacggcctgt 1800 gggcattcag tctggatcgc gaaaactgtg gaattgatca gcgttggtgg gaaagcgcgt 1860 tacaagaaag ccgggcaatt gctgtgccag gcagttttaa cgatcagttc gccgatgcag 1920 atattcgtaa ttatgcgggc aacgtctggt atcagcgcga agtctttata ccgaaaggtt 1980 gggcaggcca gcgtatcgtg ctgcgtttcg atgcggtcac tcattacggc aaagtgtggg 2040 tcaataatca ggaagtgatg gagcatcagg gcggctatac gccatttgaa gccgatgtca 2100 cgccgtatgt tattgccggg aaaagtgtac gtaagtttct gcttctacct ttgatatata 2160 tataataatt atcattaatt agtagtaata taatatttca aatatttttt tcaaaataaa 2220 agaatgtagt atatagcaat tgcttttctg tagtttataa gtgtgtatat tttaatttat 2280 aacttttcta atatatgacc aaaatttgtt gatgtgcagg tatcaccgtt tgtgtgaaca 2340 acgaactgaa ctggcagact atcccgccgg gaatggtgat taccgacgaa aacggcaaga 2400 aaaagcagtc ttacttccat gatttcttta actatgccgg aatccatcgc agcgtaatgc 2460 tctacaccac gccgaacacc tgggtggacg atatcaccgt ggtgacgcat gtcgcgcaag 2520 actgtaacca cgcgtctgtt gactggcagg tggtggccaa tggtgatgtc agcgttgaac 2580 tgcgtgatgc ggatcaacag gtggttgcaa ctggacaagg cactagcggg actttgcaag 2640 tggtgaatcc gcacctctgg caaccgggtg aaggttatct ctatgaactg tgcgtcacag 2700 ccaaaagcca gacagagtgt gatatctacc cgcttcgcgt cggcatccgg tcagtggcag 2760 tgaagggcga acagttcctg attaaccaca aaccgttcta ctttactggc tttggtcgtc 2820 atgaagatgc ggacttgcgt ggcaaaggat tcgataacgt gctgatggtg cacgaccacg 2880 cattaatgga ctggattggg gccaactcct accgtacctc gcattaccct tacgctgaag 2940 agatgctcga ctgggcagat gaacatggca tcgtggtgat tgatgaaact gctgctgtcg 3000 gctttaacct ctctttaggc attggtttcg aagcgggcaa caagccgaaa gaactgtaca 3060 gcgaagaggc agtcaacggg gaaactcagc aagcgcactt acaggcgatt aaagagctga 3120 tagcgcgtga caaaaaccac ccaagcgtgg tgatgtggag tattgccaac gaaccggata 3180 cccgtccgca aggtgcacgg gaatatttcg cgccactggc ggaagcaacg cgtaaactcg 3240 acccgacgcg tccgatcacc tgcgtcaatg taatgttctg cgacgctcac accgatacca 3300 tcagcgatct ctttgatgtg ctgtgcctga accgttatta cggatggtat gtccaaagcg 3360 gcgatttgga aacggcagag aaggtactgg aaaaagaact tctggcctgg caggagaaac 3420 tgcatcagcc gattatcatc accgaatacg gcgtggatac gttagccggg ctgcactcaa 3480 tgtacaccga catgtggagt gaagagtatc agtgtgcatg gctggatatg tatcaccgcg 3540 tctttgatcg cgtcagcgcc gtcgtcggtg aacaggtatg gaatttcgcc gattttgcga 3600 cctcgcaagg catattgcgc gttggcggta acaagaaagg gatcttcact cgcgaccgca 3660 aaccgaagtc ggcggctttt ctgctgcaaa aacgctggac tggcatgaac ttcggtgaaa 3720 aaccgcagca gggaggcaaa caatgaatca acaactctcc tggcgcacca tcgtcggcta 3780 cagcctcggg aattagatcc ccgaatttcc ccgatcgttc aaacatttgg caataaagtt 3840 tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt ctgttgaatt 3900 acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga tgggttttta 3960 tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata tagcgcgcaa 4020 actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgggaa tt 4072 

What is claimed is:
 1. DNA sequence capable of driving expression of an associated nucleotide sequence, wherein said DNA sequence comprises the nucleotide sequence depicted in SEQ ID NO:1.
 2. The DNA sequence according to claim 1, wherein said DNA sequence comprises the nucleotide sequence depicted in SEQ ID NO:2.
 3. The DNA sequence according to claim 1, wherein said DNA sequence comprises the nucleotide sequence depicted in SEQ ID NO:3.
 4. The DNA sequence according to claim 1, wherein said DNA sequence comprises the nucleotide sequence depicted in SEQ ID NO:4.
 5. The DNA sequence according to claim 1, wherein said DNA sequence comprises the nucleotide sequence depicted in SEQ ID NO:5.
 6. The DNA sequence according to claim 1, wherein said DNA sequence comprises the nucleotide sequence depicted in SEQ ID NO:6.
 7. DNA sequence which hybridizes under stringent conditions to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.
 8. The DNA sequence according to claim 7, wherein said DNA sequence comprises the nucleotide sequence depicted in SEQ ID NO:19.
 9. The DNA sequence according to claim 7, wherein said DNA sequence comprises the nucleotide sequence depicted in SEQ ID NO:20.
 10. DNA sequence comprising a consecutive stretch of at least 50 nt of SEQ ID NO:1, wherein said DNA sequence is capable of driving expression of an associated nucleotide sequence.
 11. DNA sequence according to claim 10 wherein said consecutive stretch of at least 50 nt has at least 70% sequence identity with a consecutive stretch of at least 50 nt of SEQ ID NO:1.
 12. Recombinant DNA molecule comprising a full-length transcript promoter region isolated from Cestrum yellow leaf curling virus.
 13. Recombinant DNA molecule comprising a DNA sequence according to any one of claims 1 to 11 operably linked to a nucleotide sequence of interest.
 14. The recombinant DNA molecule of claim 13, wherein the nucleotide sequence of interest comprises a coding sequence.
 15. The recombinant DNA molecule of claim 14, wherein the coding sequence encodes a desirable phenotypic trait.
 16. The recombinant DNA molecule of claim 15, wherein the coding sequence encodes a protein which confers a positive selective advantage to cells that have been transformed with said coding sequence.
 17. The recombinant DNA molecule of claim 15 or 16, wherein the coding sequence encodes a protein which confers a metabolic advantage to cells that have been transformed with said coding sequence consisting of being able to metabolize a compound, wherein said compound is mannose or xylose or a derivative or a precursor of these, or a substrate of the protein, or is capable of being metabolized by cells transformed with said coding sequence into such a substrate.
 18. The recombinant DNA molecule of claim 17, wherein said coding sequence encodes an enzyme selected from the group of xyloisomerases, phosphomanno-isomerase, mannose-6-phosphate isomerase, mannose-1-phosphate isomerase, phosphomanno mutase, mannose epimerase, mannose or xylose phosphatase, mannose-6-phosphatase, mannose-1-phosphatase and mannose or xylose permease.
 19. The recombinant molecule of claim 18, wherein the coding sequence encodes a phosphomanno isomerase.
 20. The recombinant DNA according to claim 14, wherein the coding region is non-translatable.
 21. The recombinant DNA according to claim 20, wherein the non-translatable coding region is from a viral gene.
 22. The recombinant DNA according to claim 21, wherein the viral gene is derived from TSWV, in particular from the TSWV NP gene.
 23. The recombinant DNA molecule of claim 14, wherein the coding sequence is in antisense orientation.
 24. A DNA expression vector comprising a DNA sequence according to any one of claims 1 to 11 or a recombinant DNA molecule of any one of claims 12 to
 23. 25. The DNA expression vector of claim 24, wherein said DNA expression vector is pNOV2819.
 26. The DNA expression vector of claim 24, wherein said DNA expression vector is pNOV2820.
 27. A DNA expression vector comprising a first DNA sequence according to any one of claims 1 to 11 operably linked to a nucleotide sequence of interest, and a second DNA sequence according to any one of claims 1 to 11 operably linked to a nucleotide sequence of interest.
 28. A DNA expression vector according to claim 27 capable of altering the expression of a viral genome.
 29. The DNA expression vector according to claim 28 comprising a first DNA sequence capable of expressing in a cell a sense RNA fragment of said viral genome or portion thereof and a second DNA sequence capable of expressing in a cell an antisense RNA fragment of said viral genome or portion thereof, wherein said sense RNA fragment and said antisense RNA fragment are capable of forming a double-stranded RNA.
 30. The DNA expression vector according to claim 29, wherein said virus is selected from the group consisting of tospoviruses, potyviruses, potexviruses, tobamoviruses, luteoviruses, cucumoviruses, bromoviruses, closteorviruses, tombusviruses and furoviruses.
 31. The DNA expression vector of claim 30, wherein said DNA sequences comprises a nucleotide sequence derived from a viral coat protein gene, a viral nucleocapsid protein gene, a viral replicase gene, or a viral movement protein gene or portions thereof.
 32. The DNA expression vector of claim 31, wherein said DNA sequence is derived from tomato spotted wilt virus (TSWV).
 33. The DNA expression vector of claim 32, wherein said DNA is derived from a nucleocapsid protein gene.
 34. A host cell stably transformed with a DNA sequence according to any one of claims 1 to 11 or a recombinant DNA molecule of any one of claims 12 to 23 or a DNA expression vector according to anyone of claims 24 to
 33. 35. The host cell of claim 34, wherein said host cell is a plant cell.
 36. A plant and the progeny thereof stably transformed with a DNA sequence according to any one of claims 1 to 11 or a recombinant DNA molecule of any one of claims 12 to 23 or a DNA expression vector according to anyone of claims 24 to
 33. 37. The plant of claim 36, wherein said plant is selected from the group consisting of maize, wheat, sorghum, rye, oats, turf grass, rice, barley, soybean, cotton, tobacco, sugar beet and oilseed rape.
 38. Use of the DNA sequence of any one of claims 1 to 11 to express a nucleotide sequence of interest.
 39. A method of producing a DNA sequence according to claim 1, wherein the DNA is produced by a polymerase chain reaction wherein at least one oligonucleotide used comprises a sequence of nucleotides which represents a consecutive stretch of 15 or more base pairs of SEQ ID NO:1. 